Polycistronic HIV vector constructs

ABSTRACT

The present disclosure relates to vectors comprising polynucleotide sequences that encode HIV polypeptides. In particular, the disclosure relates polycistronic vector constructs comprising sequences that encode HIV polypeptides as a single polyprotein. Compositions comprising these vectors and sequences along with methods of using these vectors and sequences are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/124,602,filed on 05 May 2005, now U.S. Pat. No. 7,622,125, which claims thebenefit of provisional Application U.S. 60/568,390, filed on 05 May2004, all of which are hereby incorporated by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, in part, with support from the U.S. Governmentunder contract NIH HIVDDT Contract # N01-AI-05396 awarded by theNational Institutes of Health. The Government may have certain rights inthis invention.

TECHNICAL FIELD

Polynucleotides encoding HIV polypeptides are described, as are uses ofthese polynucleotides and polypeptide products including formulations ofimmunogenic compositions and uses thereof. In particular, polycistronicexpression vectors are described.

BACKGROUND

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modern medicine. There is, as yet, nocure for this disease.

A great deal of information has been gathered about the HIV, however, todate an effective vaccine has not been identified. Recently, use ofpolynucleotides encoding antigenic HIV polypeptides in immunogeniccompositions has been described. See, e.g., U.S. Pat. No. 5,846,546 toHurwitz et al.; U.S. Pat. No. 5,840,313 to Vahlne et al.; U.S. Pat. No.5,876,731 to Sia et al.

Furthermore, U.S. Pat. Nos. 6,689,879; 6,602,705 and InternationalPublications WO 00/39303, WO 00/39302, WO 00/39304, WO 02/04493 describepolynucleotides encoding one or more immunogenic HIV polypeptides thatgenerate humoral and/or cellular responses in vivo.

SUMMARY

Described herein are polynucleotides encoding multiple HIV polypeptides,polypeptides encoded by these sequences, as well as methods of makingand using these polynucleotides and/or polypeptides.

In one aspect, the present invention relates to synthetic sequencesencoding multiple HIV polypeptides (e.g., sequences encoding HIV Gag,Pol, Tat, Rev and Nef polypeptides). In certain embodiments, thesequences are contained in a vector (e.g., expression cassette) andcomprise, in a 5′ to 3′ orientation, sequences as described hereinencoding Tat, Rev, Nef, Gag and Pol. Preferably, the sequences encodesingle HIV polyprotein. The polypeptides are preferably modified ascompared to wild type and may contain one or more mutations that affectone or more functions of the polypeptide. For example, protease activityof the protease region of a Pol polypeptide can be attenuated orinactivated by making the appropriate mutations to the Pol-encodingsequence. Preferably, the sequences described herein encode polypeptidesthat elicit an immunological response when administered to the subject.

In certain embodiments, the invention relates to a polynucleotidesequence encoding more than one HIV polypeptide, wherein thepolynucleotide sequence comprises a sequence having between about 85% to100% and any integer values therebetween, for example, at least about85%, preferably about 90%, more preferably about 95%, and morepreferably about 98% sequence identity to the sequences or functionalfragments thereof taught in the present specification. Further,sequences described herein may also include sequences encodingadditional polypeptides, for example, coding sequences for other viralproteins (e.g., hepatitis B or C or other HIV proteins, such as,polynucleotide sequences encoding the same or other HIV polypeptidessuch as Env, vif, vpr, tat, rev, vpu and nef); cytokines and/or othertransgenes. The polynucleotides of the present invention can be producedby recombinant techniques, synthetic techniques, or combinationsthereof.

The sequences described herein may be obtained or derived from an HIVstrain or subtype. In certain embodiments, the sequences are obtained orderived from a subtype B HIV strain, for example SF2 and/or SF162. Inother embodiments, the sequences are obtained or derived from a subtypeC HIV strain. Furthermore, one or more of the sequences encoding an HIVpolypeptide may be derived from different strains or subtypes.

In another aspect, the present invention relates to generating an immuneresponse in a subject using the sequences (e.g., contained withinexpression cassettes) of the present invention. The immune responses maybe therapeutic and/or prophylactic and may induce cellular and/orhumoral (e.g., neutralizing antibody) immune responses against HIV in asubject. In certain embodiments, an immune response is generated byadministering one or more sequences as described herein along with oneor more polypeptides.

The present invention further includes recombinant expression systemsfor use in selected host cells, wherein the recombinant expressionsystems employ one or more of the polynucleotides and expressioncassettes of the present invention. In such systems, the polynucleotidesequences are operably linked to control elements compatible withexpression in the selected host cell. Numerous expression controlelements are known to those in the art, including, but not limited to,the following: transcription promoters, transcription enhancer elements,transcription termination signals, polyadenylation sequences, sequencesfor optimization of initiation of translation, and translationtermination sequences. Exemplary transcription promoters include, butare not limited to those derived from CMV, CMV+intron A, SV40, RSV,HIV-Ltr, MMLV-ltr, and metallothionein.

In another aspect the invention includes cells comprising one or more ofthe sequences of the present invention where the polynucleotidesequences are operably linked to control elements compatible withexpression in the selected cell. In one embodiment such cells aremammalian cells. Exemplary mammalian cells include, but are not limitedto, BHK, VERO, HT1080, 293, RD, COS-7, and CHO cells. Other cells, celltypes, tissue types, etc., that may be useful in the practice of thepresent invention include, but are not limited to, those obtained fromthe following: insects (e.g., Trichoplusia ni (Tn5) and Sf9), avian(e.g., hens' cells such as hens' embryo cells (CEF cells)), bacteria,yeast, plants, antigen presenting cells (e.g., macrophage, monocytes,dendritic cells, B-cells, T-cells, stem cells, and progenitor cellsthereof), primary cells, immortalized cells, tumor-derived cells.

In another aspect, the present invention includes compositions forgenerating an immunological response, where the composition typicallycomprises at least one of the expression cassettes of the presentinvention and may, for example, contain combinations of expressioncassettes such as one or more expression cassettes. Such compositionsmay further contain an adjuvant or adjuvants. The compositions may alsocontain one or more HIV polypeptides. The HIV polypeptides maycorrespond to the polypeptides encoded by the expression cassette(s) inthe composition, or may be different from those encoded by theexpression cassettes. In compositions containing both expressioncassettes (or polynucleotides of the present invention) andpolypeptides, various expression cassettes of the present invention canbe mixed and/or matched with various HIV polypeptides described herein.

In another aspect the present invention includes methods of immunizationof a subject. In the method any of the above-described compositions areinto the subject under conditions that are compatible with expression ofthe expression cassette(s) in the subject. In one embodiment, theexpression cassettes (or polynucleotides of the present invention) canbe introduced using a gene delivery vector. The gene delivery vectorcan, for example, be a non-viral vector or a viral vector. Exemplaryviral vectors include, but are not limited to eukaryotic layered vectorinitiation systems, Sindbis-virus (or other alphavirus) derived vectors,retroviral vectors, and lentiviral vectors. Other exemplary vectorsinclude, but are not limited to, pCMVKm2, pCMV6a, pCMV-link, andpCMVPLEdhfr. Compositions useful for generating an immunologicalresponse can also be delivered using a particulate carrier (e.g., PLG orCTAB-PLG microparticles). Further, such compositions can be coated on,for example, gold or tungsten particles and the coated particlesdelivered to the subject using, for example, a gene gun. Thecompositions can also be formulated as liposomes. In one embodiment ofthis method, the subject is a mammal and can, for example, be a human.

The polynucleotides of the present invention may be employed singly orin combination.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1B depict the nucleotide sequence of a construct designatedTRNgagCpolIna (SEQ ID NO:1). The construct includes synthetic sequencesencoding an HIV polyprotein including HIV Tat, Rev, Nef, Gag and Polpolypeptides. The protease function of Pol has been inactivated. Thesequences encoding the HIV polyprotein are ordered, in a 5′ to 3′direction, Tat-, Rev-, Nef-, Gag-, Pol-encoding sequences. In addition,the construct includes HIV protease cleavage sites (ATIM/MQR) encoded byresidues 1294 to 1314, 2443 to 2463, and 2878 to 2898 of FIG. 1A.

FIGS. 2A to 2B depict the nucleotide sequence of a construct designatedgagCpolInaTRN (SEQ ID NO:2). The construct includes synthetic sequencesencoding an HIV polyprotein including HIV Gag, Pol, Tat, Rev, Nef,polypeptides. The protease function of Pol has been inactivated. Thesequences encoding the HIV polyprotein are ordered, in a 5′ to 3′direction, Gag-, Pol-, Tat-, Rev-, Nef-encoding sequences. In addition,the construct includes HIV protease cleavage sites (ATIM/MQR) encoded byresidues 1,138 to 1,158 and 1,573 to 1,593.

FIG. 3, panels A and B, are reproductions of gels showing Western Blotanalysis of protein levels in lysate of cells transfected with variousHIV polypeptide-encoding constructs. Panel A depicts results obtained 48hours post-transfection. Panel B depicts results obtained 72 hourspost-transfection. In both panels, the lane designated “M” shows themarker. Lane 1 is shows protein levels in cells transfected withconstructs encoding Gag. Lane 2 shows protein levels in cellstransfected with constructs encoding Gag and Pol. The protease functionof Pol has been inactivated. Lane 3 shows protein levels in cellstransfected with constructs comprising, in 5′ to 3′ orientation,sequences encoding, Gag, Pol (inactivated), Tat, Rev and Nef. Lane 4shows protein levels in lysates of cells transfected with constructscomprising, in a 5′ to 3′ orientation, sequences encoding, Tat, Rev,Nef, Gag, and inactivated Pol. Lane 5 shows protein levels in cellstransfected with constructs encoding Gag and Pol. The protease functionof Pol has been attenuated. Lane 6 shows protein levels in cellstransfected with constructs comprising, in 5′ to 3′ orientation,sequences encoding, Gag, Pol (attenuated), Tat, Rev and Nef. Lane 7shows protein levels in lysates of cells transfected with constructscomprising, in a 5′ to 3′ orientation, sequences encoding, Tat, Rev,Nef, Gag, and attenuated Pol. Lane 8 shows protein levels from cellstransfected with a construct encoding p2Pol. Lane 9 is a mock lysatecontrol.

FIG. 4, panels A and B, are reproductions of gels showing Western Blotanalysis of protein levels in supernatants of cells transfected withvarious HIV polypeptide-encoding constructs. Lane designations are thesame as in FIG. 3, except the protein levels are measured fromsupernatant not lysate.

FIG. 5, panels A and B, are reproductions of gels showing Western Blotanalysis of protein levels in lysates of cells transfected with variousHIV polypeptide-encoding constructs. Lanes designated “72,” “48,” or“24” refer to the time point (in hours) at which the experiments wereconducted. Lanes designated “1” show results from a construct(pCMVKM2TRN5′gCpIna) encoding a Gag, Pol (inactivated), Tat, rev and nefpolyprotein, in which Tat, rev and nef sequences are 5′ to the Gag andPol encoding sequences. Lanes designated with a “2” show results from aconstruct (pCMVKM2gCpInaTRN3′) encoding a Gag, Pol (inactivated), Tat,rev and nef polyprotein, in which Tat, rev and nef sequences are 3′ tothe Gag and Pol encoding sequences. Lanes designated with a “3” showresults from a construct (pCMVKM2gCpIna) encoding a Gag and Pol(inactivated) polyprotein.

FIG. 6, panels A and B, are reproductions of gels showing Western Blotanalysis of protein levels in supernatants of cells transfected withvarious HIV polypeptide-encoding constructs. Lane designations are thesame as in FIG. 5, except the protein levels are measured fromsupernatant not lysate.

FIG. 7 is a graph depicting results of Gag ELISAs of mouse bleedsfollowing administration to the animals of the constructs listed.Constructs pCMV-gagCpolIna, pCMV-TRNgagCpolIna and pCMV-gagCpolInaTRNrefer to polycistronic constructs expressing, respectively, a Gag andinactivated Pol polyprotein, a Tat, rev, nef, Gag, and inactivated Polpolyprotein, and a Gag, inactivate Pol, Tat, rev, nef polyprotein.Constructs pCMV-TNR+gagCpolIna and pCMV-Tat+gagCpolIna refer toconstructs which express Gag and inactivated Pol separately from eithera Tat, rev, nef polyprotein (TRN) or tat.

FIG. 8 is a graph depicting results of cellular immune responses (% ofIFN-gamma secreting CD8 T cells), as measured by intracellular cytokinestaining (FACS-flow cytometry) assay, to the same constructs as shown inFIG. 7.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification, the singular forms “a,” “an” and “the”include plural references unless the content clearly dictates otherwise.Thus, for example, reference to “an antigen” includes a mixture of twoor more such agents.

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Synthetic” sequences, as used herein, refers to HIVpolypeptide-encoding polynucleotides whose expression has been modifiedas described herein, for example, by codon substitution, alteredactivities, and/or inactivation of inhibitory sequences. “Wild-type” or“native” sequences, as used herein, refers to polypeptide encodingsequences that are essentially as they are found in nature, e.g., Gag,Pol, Vif, Vpr, Tat, Rev, Vpu, Env and/or Nef encoding sequences as foundin HIV isolates, e.g., SF162, SF2, AF110965, AF110967, AF110968,AF110975, 8_(—)5_TV1_C.ZA, 8_(—)2_TV1_C.ZA or 12-5_(—)1_TV2_C.ZA.

Thus, the term “Pol” refers to one or more of the followingpolypeptides: polymerase (p6Pol); protease (prot); reverse transcriptase(p66RT or RT); RNAseH (p15RNAseH); and/or integrase (p31 Int or Int).Identification of gene regions for any selected HIV isolate can beperformed by one of ordinary skill in the art based on the teachingspresented herein and the information known in the art, for example, byperforming alignments relative to other known HIV isolates, for example,Subtype β isolates with gene regions (e.g., SF2, GenBank Accessionnumber K02007; SF162, GenBank Accession Number M38428, both hereinincorporated by reference) and Subtype C isolates with gene regions(e.g., GenBank Accession Number AF110965 and GenBank Accession NumberAF110975, both herein incorporated by reference).

The term “HIV polypeptide” refers to any amino acid sequence thatexhibits sequence homology to native HIV polypeptides (e.g., Gag, Env,Prot, Pol, RT, Int, vif, vpr, vpu, tat, rev, nef and/or combinationsthereof) and/or which is functional. Non-limiting examples of functionsthat may be exhibited by HIV polypeptides include, use as immunogens(e.g., to generate a humoral and/or cellular immune response, includingimmune responses that are specific to the HIV polypeptide(s)), use indiagnostics (e.g., bound by suitable antibodies for use in ELISAs orother immunoassays) and/or polypeptides which exhibit one or morebiological activities associated with the wild type or synthetic HIVpolypeptide. For example, as used herein, the term “Gag polypeptide” mayrefer to a polypeptide that is bound by one or more anti-Gag antibodies;elicits a humoral and/or cellular immune response; and/or exhibits theability to form particles.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fingi, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein. Similarly, an oligonucleotide or polynucleotide thatexpresses an antigen or antigenic determinant in vivo, such as in genetherapy and DNA immunization applications, is also included in thedefinition of antigen herein.

For purposes of the present invention, immunogens can be derived fromany of several known viruses, bacteria, parasites and fungi, asdescribed more fully below, for example immunogens derived from an HIV.Furthermore, for purposes of the present invention, an “immunogen”refers to a protein that includes modifications, such as deletions,additions and substitutions (generally conservative in nature), to thenative sequence, so long as the protein maintains the ability to elicitan immunological response, as defined herein. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts that produce the antigens. By“immunogenic fragment” is meant a fragment of an HIV polypeptide thatincludes one or more epitopes and thus elicits one or more of theimmunological responses described herein. Such fragments can beidentified by, e.g., concurrently synthesizing large numbers of peptideson solid supports, the peptides corresponding to portions of the proteinmolecule, and reacting the peptides with antibodies while the peptidesare still attached to the supports. Such techniques are known in the artand described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984)Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec.Immunol. 23:709-715, all incorporated herein by reference in theirentireties.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular immunogen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique)(reviewedby McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med. 187 (9)1367-1371, 1998; Mcheyzer-Willians, M. G., et al, Immunol. Rev.150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

Thus, an immunological response as used herein may be one thatstimulates the production of antibodies (e.g., neutralizing antibodiesthat block bacterial toxins and pathogens such as viruses entering cellsand replicating by binding to toxins and pathogens, typically protectingcells from infection and destruction). The antigen of interest may alsoelicit production of CTLs. Hence, an immunological response may includeone or more of the following effects: the production of antibodies byB-cells; and/or the activation of suppressor T-cells and/or δγ T-cellsdirected specifically to an antigen or antigens present in thecomposition or vaccine of interest. These responses may serve toneutralize infectivity, and/or mediate antibody-complement, or antibodydependent cell cytotoxicity (ADCC) to provide protection to an immunizedhost. Such responses can be determined using standard immunoassays andneutralization assays, well known in the art. (See, e.g., Montefiori etal. (1988) J. Clin Microbiol. 26:231-235; Dreyer et al. (1999) AIDS ResHum Retroviruses (1999) 15(17):1563-1571).

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest. The immunogeniccomposition can be introduced directly into a recipient subject, such asby injection, inhalation, oral, intranasal and mucosal (e.g.,intra-rectally or intra-vaginally) administration.

By “subunit vaccine” is meant a vaccine composition that includes one ormore selected antigens but not all antigens, derived from or homologousto, an antigen from a pathogen of interest such as from a virus,bacterium, parasite or fungus. Such a composition is substantially freeof intact pathogen cells or pathogenic particles, or the lysate of suchcells or particles. Thus, a “subunit vaccine” can be prepared from atleast partially purified (preferably substantially purified) immunogenicpolypeptides from the pathogen, or analogs thereof. The method ofobtaining an antigen included in the subunit vaccine can thus includestandard purification techniques, recombinant production, or syntheticproduction.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence that “encodes” a selected polypeptideis a nucleic acid molecule that is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences (or “controlelements”). The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxy) terminus. A coding sequence can include, but is notlimited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNAsequences from viral or prokaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence such as a stop codon maybe located 3′ to the coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences.

A “polynucleotide coding sequence” or a sequence that “encodes” aselected polypeptide, is a nucleic acid molecule that is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vivo when placed under the control of appropriate regulatorysequences (or “control elements”). The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. Exemplary codingsequences are the modified viral polypeptide-coding sequences of thepresent invention. A transcription termination sequence may be located3′ to the coding sequence. Typical “control elements”, include, but arenot limited to, transcription regulators, such as promoters,transcription enhancer elements, transcription termination signals, andpolyadenylation sequences; and translation regulators, such as sequencesfor optimization of initiation of translation, e.g., Shine-Dalgarno(ribosome binding site) sequences, Kozak sequences (i.e., sequences forthe optimization of translation, located, for example, 5′ to the codingsequence), leader sequences (heterologous or native), translationinitiation codon (e.g., ATG), and translation termination sequences. Incertain embodiments, one or more translation regulation or initiationsequences (e.g., the leader sequence) are derived from wild-typetranslation initiation sequences, i.e., sequences that regulatetranslation of the coding region in their native state. Wild-type leadersequences that have been modified, using the methods described herein,also find use in the present invention. Native or modified leadersequences can be from any source, for example other strains, variantsand/or subtypes of HIV or non-HIV sources (e.g., tpa leader sequenceexemplified herein). Promoters can include inducible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris induced by an analyte, cofactor, regulatory protein, etc.),repressible promoters (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), and constitutive promoters.

A “nucleic acid” molecule can include, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA,genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting prokaryotic microorganisms or eukaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, cl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Genetics Computer Group (Madison, Wis.) intheir BestFit utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). Other equally suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity.” Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:www.ncbi.nlm.gov/cgi-bin/BLAST.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence, exemplary preferred SmithWaterman based parameters are presented above. For example, the searchparameters may vary based on the size of the sequence in question. Thus,for the polynucleotide sequences of the present invention the length ofthe polynucleotide sequence disclosed herein is searched against aselected database and compared to sequences of essentially the samelength to determine percent identity. For example, a representativeembodiment of the present invention would include an isolatedpolynucleotide having X contiguous nucleotides, wherein (i) the Xcontiguous nucleotides have at least about a selected level of percentidentity relative to Y contiguous nucleotides of the sequences describedherein, and (ii) for search purposes X equals Y, wherein Y is a selectedreference polynucleotide of defined length.

The sequences of the present invention can include fragments of thesequences, for example, from about 15 nucleotides up to the number ofnucleotides present in the full-length sequences described herein (e.g.,see the Sequence Listing, Figures, and claims), including all integervalues falling within the above-described range. For example, fragmentsof the polynucleotide sequences of the present invention may be 30-60nucleotides, 60-120 nucleotides, 120-240 nucleotides, 240-480nucleotides, 480-1000 nucleotides, and all integer values therebetween.

The synthetic polynucleotides of the present invention include relatedpolynucleotide sequences having about 80% to 100%, greater than 80-85%,preferably greater than 90-92%, more preferably greater than 92-95%,more preferably greater than 95%, and most preferably greater than 98%up to 100% (including all integer values falling within these describedranges) sequence identity to the synthetic polynucleotide sequencesdisclosed herein (for example, to the claimed sequences or othersequences of the present invention) when the sequences of the presentinvention are used as the query sequence against, for example, adatabase of sequences.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supraor Ausubel et al., supra). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al., supraor Ausubel et al., supra).

A first polynucleotide is “derived from” second polynucleotide if it hasthe same or substantially the same basepair sequence as a region of thesecond polynucleotide, its cDNA, complements thereof, or if it displayssequence identity as described above.

A first polypeptide is “derived from” a second polypeptide if it is (i)encoded by a first polynucleotide derived from a second polynucleotide,or (ii) displays sequence identity to the second polypeptides asdescribed above.

Generally, a viral polypeptide: is “derived from” a particularpolypeptide of a virus (viral polypeptide) if it is (i) encoded by anopen reading frame of a polynucleotide of that virus (viralpolynucleotide), or (ii) displays sequence identity to polypeptides ofthat virus as described above.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences that areimmunologically identifiable with a polypeptide encoded by the sequence.Further, polyproteins can be constructed by fusing in-frame two or morepolynucleotide sequences encoding polypeptide or peptide products.Further, polycistronic coding sequences may be produced by placing twoor more polynucleotide sequences encoding polypeptide products adjacenteach other, typically under the control of one promoter, wherein eachpolypeptide coding sequence may be modified to include sequences forinternal ribosome binding sites.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof that is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA of interest into a host cell. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from alphaviruses, pox virusesand vaccinia viruses. When used for immunization, such gene deliveryexpression vectors may be referred to as vaccines or vaccine vectors.

“T lymphocytes” or “T cells” are non-antibody producing lymphocytes thatconstitute a part of the cell-mediated arm of the immune system. T cellsarise from immature lymphocytes that migrate from the bone marrow to thethymus, where they undergo a maturation process under the direction ofthymic hormones. Here, the mature lymphocytes rapidly divide increasingto very large numbers. The maturing T cells become immunocompetent basedon their ability to recognize and bind a specific antigen. Activation ofimmunocompetent T cells is triggered when an antigen binds to thelymphocyte's surface receptors.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

A “vector” is capable of transferring gene sequences to target cells(e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a gene of interest and which can transfergene sequences to target cells. Thus, the term includes cloning andexpression vehicles, as well as viral vectors.

Transfer of a “suicide gene” (e.g., a drug-susceptibility gene) to atarget cell renders the cell sensitive to compounds or compositions thatare relatively nontoxic to normal cells. Moolten, F. L. (1994) CancerGene Ther. 1:279-287. Examples of suicide genes are thymidine kinase ofherpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996)Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al.(1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosinedeaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells thatexpress these genes are rendered sensitive to the effects of therelatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase)or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992)Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA91:8302-8306.

A “selectable marker” or “reporter marker” refers to a nucleotidesequence included in a gene transfer vector that has no therapeuticactivity, but rather is included to allow for simpler preparation,manufacturing, characterization or testing of the gene transfer vector.

A “specific binding agent” refers to a member of a specific binding pairof molecules wherein one of the molecules specifically binds to thesecond molecule through chemical and/or physical means. One example of aspecific binding agent is an antibody directed against a selectedantigen.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (I) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

By “co-administration” is meant administration of more than onecomposition or molecule. Thus, co-administration includes concurrentadministration or sequentially administration (in any order), via thesame or different routes of administration. Non-limiting examples ofco-administration regimes include, co-administration of nucleic acid andpolypeptide; co-administration of different nucleic acids (e.g.,different expression cassettes as described herein and/or different genedelivery vectors); and co-administration of different polypeptides(e.g., different HIV polypeptides and/or different adjuvants). The termalso encompasses multiple administrations of one of the co-administeredmolecules or compositions (e.g., multiple administrations of one or moreof the polynucleotides and/or expression cassettes described hereinfollowed by one or more administrations of a polypeptide-containingcomposition). In cases where the molecules or compositions are deliveredsequentially, the time between each administration can be readilydetermined by one of skill in the art in view of the teachings herein.

“Lentiviral vector”, and “recombinant lentiviral vector” refer to anucleic acid construct that carries, and within certain embodiments, iscapable of directing the expression of a nucleic acid molecule ofinterest. The lentiviral vector include at least one transcriptionalpromoter/enhancer or locus defining element(s), or other elements whichcontrol gene expression by other means such as alternate splicing,nuclear RNA export, post-translational modification of messenger, orpost-transcriptional modification of protein. Such vector constructsmust also include a packaging signal, long terminal repeats (LTRS) orportion thereof, and positive and negative strand primer binding sitesappropriate to the retrovirus used (if these are not already present inthe retroviral vector). Optionally, the recombinant lentiviral vectormay also include a signal that directs polyadenylation, selectablemarkers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, aswell as one or more restriction sites and a translation terminationsequence. By way of example, such vectors typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second strand DNAsynthesis, and a 3′LTR or a portion thereof “Lentiviral vector particle”as utilized within the present invention refers to a lentivirus thatcarries at least one gene of interest. The retrovirus may also contain aselectable marker. The recombinant lentivirus is capable of reversetranscribing its genetic material (RNA) into DNA and incorporating thisgenetic material into a host cell's DNA upon infection. Lentiviralvector particles may have a lentiviral envelope, a non-lentiviralenvelope (e.g., an ampho or VSV-G envelope), or a chimeric envelope. An“alphavirus vector” refers to a nucleic acid construct that carries, andwithin certain embodiments, is capable of directing the expression of anucleic acid molecule of interest. Alphavirus vectors may be utilized inseveral formats, including DNA, RNA, and recombinant replicon particles.Such replicon vectors have been derived from alphaviruses that include,for example, Sindbis virus, Semliki Forest virus, and/or Venezuelanequine encephalitis virus. See, e.g., U.S. Pat. Nos. 5,789,245;5,814,482; and 6,376,235 and WO 02/099035. Chimeric alphavirus vectorsare described, for example, in U.S. Patent Publications 20030232324 and20030148262. The terms “alphavirus RNA replicon vector”, “RNA repliconvector”, “replicon vector” or “replicon” refer to an RNA molecule thatis capable of directing its own amplification or self-replication invivo, within a target cell. To direct its own amplification, the RNAmolecule should encode the polymerase(s) necessary to catalyze RNAamplification (e.g., alphavirus nonstructural proteins nsP1, nsP2, nsP3,nsP4) and also contain cis RNA sequences required for replication whichare recognized and utilized by the encoded polymerase(s). An alphavirusRNA vector replicon typically contains following ordered elements: 5′viral or cellular sequences required for nonstructural protein-mediatedamplification (may also be referred to as 5′ CSE, or 5′ cis replicationsequence, or 5′ viral sequences required in cis for replication, or 5′sequence which is capable of initiating transcription of an alphavirus),sequences which, when expressed, code for biologically active alphavirusnonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), and 3′ viral orcellular sequences required for nonstructural protein-mediatedamplification (may also be referred as 3′ CSE, or 3′ viral sequencesrequired in cis for replication, or an alphavirus RNA polymeraserecognition sequence). The alphavirus RNA vector replicon also shouldcontain a means to express one or more heterologous sequence(s), such asfor example, an IRES or a viral (e.g., alphaviral) subgenomic promoter(e.g., junction region promoter) which may, in certain embodiments, bemodified in order to increase or reduce viral transcription of thesubgenomic fragment, or to decrease homology with defective helper orstructural protein expression cassettes, and one or more heterologoussequence(s) to be expressed. When used as vectors, the replicons willalso contain additional sequences, for example, one or more heterologoussequence(s) encoding one or more polypeptides (e.g., a protein-encodinggene or a 3′ proximal gene) and/or a polyadenylate tract.

“Nucleic acid expression vector” or “Expression cassette” refers to anassembly that is capable of directing the expression of a sequence orgene of interest. The nucleic acid expression vector typically includesa promoter that is operably linked to the sequences or gene(s) ofinterest. Other control elements may be present as well. Expressioncassettes described herein may be contained within a plasmid construct.In addition to the components of the expression cassette, the plasmidconstruct may also include a bacterial origin of replication, one ormore selectable markers, a signal which allows the plasmid construct toexist as single-stranded DNA (e.g., a M113 origin of replication), amultiple cloning site, and a “mammalian” origin of replication (e.g., aSV40 or adenovirus origin of replication).

“Packaging cell” refers to a cell that contains those elements necessaryfor production of infectious recombinant viral that are lacking in arecombinant viral vector. Typically, such packaging cells contain one ormore expression cassettes that are capable of expressing proteinsencoded by the sequences described herein.

“Producer cell” or “vector producing cell” refers to a cell thatcontains all elements necessary for production of recombinant viralvector particles.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

2. General Overview

Numerous studies conducted in humans and animals have clearlydemonstrated that, in order to be effective, HIV vaccines must includesufficient amounts of HIV proteins. Typically, the levels of expressionrequired for a robust immune response are higher than those producedfrom wild-type sequences. Consequently, we have previously developedconstructs in which the sequences are modified to improve expressionover wild type and provide robust immune responses. See, e.g., U.S. Pat.No. 6,602,705; and International Publications WO 02/00250; WO 02/04493;WO 03/004620; WO 03/004657; and WO 03/020876.

Described herein are sequences that express HIV proteins at higherlevels than wild type sequences. High levels of protein expression are adesirable starting point for the development of immunogeniccompositions. In particular, polycistronic vectors comprising sequencesencoding various HIV polypeptides as a single polyprotein are described,wherein expression of the HIV polypeptides is improved as compared towild type.

3. The HIV Genome

The HIV genome and various polypeptide-encoding regions are known. Forexample, GenBank Accession No. K02007 describes various regions ofHIV_(sF2) (“SF2”). It will be readily apparent to one of ordinary skillin the art in view of the teachings of the present disclosure how todetermine corresponding regions in other HIV strains or variants (e.g.,isolates HIV_(IIIb), HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV), HIV_(LAI),HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diversesubtypes (e.g., subtypes, A through G, and O), HIV-2 strains and diversesubtypes (e.g., HIV-2_(UC1) and HIV-2_(UC2)), and simianimmunodeficiency virus (SIV). (See, e.g., Virology, 3rd Edition (W. K.Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D.M. Knipe, eds. 1991); Virology, 3rd Edition (Fields, B N, DM Knipe, P MHowley, Editors, 1996, Lippincott-Raven, Philadelphia, Pa.; for adescription of these and other related viruses), using for example,sequence comparison programs (e.g., BLAST and others described herein)or identification and alignment of structural features (e.g., a programsuch as the “ALB” program described herein that can identify the variousregions).

4. Synthetic Sequences

One aspect of the present invention is the generation of syntheticsequences (modified as compared to wild type) encoding HIV-1polypeptides, and related sequences. When incorporated into anexpression vector, the sequences exhibit improved expression relative tothe corresponding wild-type sequences. In addition, when the sequencesincorporated into the vector are ordered such that Gag- and Pol-encodingsequences are 3′ to sequences encoding Tat, Rev and Nef, expression inboth lysate and supernatant is further improved as compared toindividual synthetic sequences and to synthetic sequences ordered withTat, Rev and Nef sequences 3′ to Gag and Pol.

The sequences described herein were prepared essentially as described inU.S. Pat. No. 6,602,705. Briefly, HIV-1 codon usage pattern was modifiedso that the resulting nucleic acid coding sequence was comparable tocodon usage found in highly expressed human genes. In addition,inhibitory (or instability) elements (INS) located within the codingsequences of, for example, the Gag and/or protease coding sequences wereinactivated or attenuated, for example by introducing multiple pointmutations that did not alter the reading frame of the encoded proteins.Furthermore, for some genes the coding sequence has been altered suchthat the polynucleotide coding sequence encodes a gene product that isinactive or non-functional (e.g., inactivated polymerase, protease, tat,rev, nef, vif, vpr, and/or vpu gene products). Example 1 describes someexemplary mutations.

Synthetic expression cassettes, derived from HIV Type B codingsequences, exemplified herein include, but are not limited to, thoseshown in FIGS. 1 and 2. “Gag-complete” or “Gagc” refers to in-framepolyproteins comprising, e.g., Gag and pol, wherein the p6 portion ofGag is present.

Additional polynucleotide sequences that may be employed in some aspectsof the present invention have been described in U.S. Pat. No. 6,689,879;U.S. Pat. No. 6,602,705; WO 00/39302, WO 00/39303, WO 00/39304, and WO02/04493, all of which are herein incorporated by reference in theirentireties.

Further, the synthetic sequences of the present invention includerelated polynucleotide sequences having greater than 85%, preferablygreater than 90%, more preferably greater than 95%, and most preferablygreater than 98% sequence identity to the synthetic polynucleotidesequences disclosed herein. Sequences exhibiting the requisite homologymay be generated, for example, by gene shuffling techniques as describedfor example in U.S. Pat. Nos. 6,323,030; 6,444,468; 6,420,175; and6,413,774, incorporated herein in their entireties by reference.

The synthetic coding sequences are assembled by methods known in theart, for example by companies such as the Midland Certified ReagentCompany (Midland, Tex.).

As noted above, the synthetic sequences described herein are modifiedfrom wild type codon usage to codon usage more typical in humans.Additional modifications include, but are not limited to, addition ofcleavage sites, leader sequences or the like; addition of othersequences (HIV or non-HIV) and/or introduction of mutations into one ormore of the sequences such that non-functional variants were created.

For instance, the protease activity of Pol is preferably attenuated orinactivated. In other embodiments, the integrase and/or RNase H activityof Pol is attenuated or inactivated. Table A sets forth exemplarymutations affecting the activity of several HIV genes. All referencescited are herein incorporated by reference.

TABLE A Gene “Region” Exemplary Mutations Pol prot Att = Reducedactivity by attenuation of Protease (Thr26Ser) (e.g., Konvalinka et al.,1995, J Virol 69: 7180-86) Ina = Mutated Protease, nonfunctional enzyme(Asp25Ala)(e.g., Konvalinka et al., 1995, J Virol 69: 7180-86) RT YM =Deletion of catalytic center (YMDD_AP; SEQ ID NO: 7) (e.g.,Biochemistry, 1995, 34, 5351, Patel et. al.) WM = Deletion of primergrip region (WMGY_PI; SEQ ID NO: 8)) (e.g., J Biol Chem, 272, 17, 11157,Palaniappan, et. al., 1997) RNase no direct mutations, RnaseH isaffected by “WM” mutation in RT Integrase 1) Mutation of HHCC domain,Cys40Ala (e.g., Wiskerchen et. al., 1995, J Virol, 69: 376). 2.)Inactivation catalytic center, Asp64Ala, Asp116Ala, Glu152Ala (e.g.,Wiskerchen et. al., 1995, J Virol, 69: 376). 3) Inactivation of minimalDNA binding domain (MDBD), deletion of Trp235(e.g., Ishikawa et. al.,1999, J Virol, 73: 4475). Constructs int. opt. mut. SF2 and int. opt.mut_C (South Africa TV1) both contain all these mutations (1, 2, and 3)Tat Mutants of Tat in transactivation domain (e.g., Caputo et al., 1996,Gene Ther. 3: 235) cys22 mutant (Cys22Gly) = TatC22 cys37 mutant(Cys37Ser) = TatC37 cys22/37 double mutant = TatC22/37 Rev Mutations inRev domains (e.g., Thomas et al., 1998, J Virol. 72: 2935-44) Mutationin RNA binding-nuclear localization ArgArg38, 39AspLeu = M5 Mutation inactivation domain LeuGlu78, 79AspLeu = M10 Nef Mutations ofmyristoyilation signal and in oligomerization domain: 1. Single pointmutation myristoyilation signal: Gly-to-Ala = -Myr 2. Deletion ofN-terminal first 18 (sub-type B, e.g., SF162) or 19 (sub-type C, e.g.,South Africa clones) amino acids: -Myr18 or - Myr19 (respectively)(e.g., Peng and Robert-Guroff, 2001, Immunol Letters 78: 195-200) Singlepoint mutation oligomerization: (e.g., Liu et al., 2000, J Virol 74:5310-19) Asp125Gly (sub B SF162) or Asp124Gly (sub C South Africaclones) Mutations affecting (1) infectivity (replication) of HIV-virionsand/or (2) CD4 down regulation. (e.g., Lundquist et al. (2002) J Virol.76(9): 4625-33)

The sequences may include a sequence that encodes the first 6 aminoacids of the integrase polypeptide. This 6 amino acid region is believedto provide a cleavage recognition site recognized by HIV protease (see,e.g., McComack et al. (1997) FEBS Letts 414:84-88). Constructs mayinclude a multiple cloning site (MCS) for insertion of one or moretransgenes, typically at the 3′ end of the construct. In addition, acassette encoding a catalytic center epitope derived from the catalyticcenter in RT is typically included 3′ of the sequence encoding 6 aminoacids of integrase. This cassette encodes Ile178 through Serine 191 ofRT and may be added to keep this well conserved region as a possible CTLepitope. Further, the constructs contain an insertion mutations topreserve the reading frame. (see, e.g., Park et al. (1991) J. Virol.65:5111).

The HIV polypeptide-encoding expression cassettes described herein mayalso contain one or more further sequences encoding, for example, one ormore transgenes. Further sequences (e.g., transgenes) useful in thepractice of the present invention include, but are not limited to,further sequences encoding further viral epitopes/antigens, includingbut not limited to,

HIV antigens (e.g., derived from one or more HIV isolate, includingHIV-1 or HIV-2 strain antigens, such as gag (p24gag and p5gag), env(gp160 and gp41), pol, tat, nef, rev, vpu, miniproteins, (preferably p55gag and gp140v delete) and antigens from the isolates HIV_(IIIb),HIV_(SF2), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235), HIV-1_(US4),HIV-2; simian immunodeficiency virus (SIV) among others;

Hepatitis A virus, such as inactivated virus;

Hepatitis B virus (HBV) antigens, such as the surface and/or coreantigens (sAg), as well as the presurface sequences, pre-S1 and pre-S2(formerly called pre-S), as well as combinations of the above, such assAg/pre-S1, sAg/pre-S2, sAg/pre-S1/pre-S2, and pre-S1/pre-S2, (see,e.g., “HBV Vaccines-Human Vaccines and Vaccination, pp. 159-176; andU.S. Pat. Nos. 4,722,840, 5,098,704, 5,324,513; Beames et al., J. Virol.(1995) 69:6833-6838, Birnbaum et al., J. Virol. (1990) 64:3319-3330; andZhou et al., J. Virol. (1991) 65:5457-5464);

Hepatitis C virus (HCV) antigens (e.g., E1, E2, E1/E2; see, Houghton etal., Hepatology (1991); Houghton, M., et al., U.S. Pat. No. 5,714,596,issued Feb. 3, 1998; Houghton, M., et al., U.S. Pat. No. 5,712,088,issued Jan. 27, 1998; Houghton, M., et al., U.S. Pat. No. 5,683,864,issued Nov. 4, 1997; Weiner, A. J., et al., U.S. Pat. No. 5,728,520,issued Mar. 17, 1998; Weiner, A. J., et al., U.S. Pat. No. 5,766,845,issued Jun. 16, 1998; Weiner, A. J., et al., U.S. Pat. No. 5,670,152,issued Sep. 23, 1997; all herein incorporated by reference; NS345polyprotein, NS 345-core polyprotein, core, and/or peptides from thenonstructural regions (WO 89/04669; WO 90/11089; and WO 90/14436);

Delta hepatitis virus (HDV) antigens, particularly δ-antigen from HDV(see, e.g., U.S. Pat. No. 5,378,814);

Hepatitis E virus (HEV) antigens;

Hepatitis G virus (HGV) antigens;

Varcicella zoster virus antigens, particularly antigens derived fromvaricella zoster virus (VZV) (J. Gen. Virol. (11986) 67:1759);

Epstein-Barr virus antigens, particularly antigens derived from EBV(Baer et al., Nature (1984) 310:207);

Cytomegalovirus (CMV) antigens, including gB and gH (Cytomegaloviruses(J. K. McDougall, ed., Springer-Verlag 1990) pp. 125-169);

Herpes simplex virus antigens including antigens from HSV-1 or HSV-2strains and glycoproteins gB, gD and gH (McGeoch et al., J. Gen. Virol.(1988) 69:1531 and U.S. Pat. No. 5,171,568);

Human Herpes Virus antigens, such as antigens derived from other humanherpesviruses such as HHV6 and HHV7; and

HPV antigens, including antigens associated with or derived from humanpapillomavirus (HPV), for example, one or more of E1-E7, L1, L2, andfusions thereof, particularly the compositions of the invention mayinclude a virus-like particle (VLP) comprising the L1 major capsidprotein, more particular still, the HPV antigens are protective againstone or more of HPV serotypes 6, 11, 16 and/or 18;

Influenza, including whole viral particles (attenuated), split, orsubunit comprising hemagglutinin (HA) and/or neuraminidase (NA) surfaceproteins, the influenza antigens may be derived from chicken embryos orpropogated on cell culture, and/or the influenza antigens are derivedfrom influenza type A, B, and/or C, among others;

Respiratory syncytial virus (RSV) antigens including the F protein ofthe A2 strain of RSV (J Gen Virol. 2004 November; 85 (Pt 11):3229)and/or G glycoprotein;

Parainfluenza virus (PIV) antigens including PIV type 1, 2, and 3,preferably containing hemagglutinin, neuraminidase and/or fusionglycoproteins;

Poliovirus antigens including antigens from a family of picornaviridae,preferably poliovirus antigens such as OPV or, preferably IPV;

Measles antigens including split measles virus (MV) antigen optionallycombined with the Protollin and or antigens present in MMR vaccine;

Mumps antigens including antigens present in MMR vaccine;

Rubella antigens including antigens present in MMR vaccine as well asother antigens from Togaviridae, including dengue virus;

Rabies such as lyophilized inactivated virus (RabAvert™);

Flaviviridae viruses such as (and antigens derived therefrom) yellowfever virus, Japanese encephalitis virus, dengue virus (types 1, 2, 3,or 4), tick borne encephalitis virus, and West Nile virus;

Caliciviridae antigens therefrom;

Rotavirus including VP4, VP5, VP6, VP7, VP8 proteins (Protein ExprPurif. 2004 December; 38(2):205) and/or NSP4;

Pestivirus antigens such as antigens from classical porcine fever virus,bovine viral diarrhoea virus, and/or border disease virus;

Parvovirus such as parvovirus B19;

Coronavirus antigens including SARS virus antigens, particularly spikeprotein or proteases therefrom, as well as antigens included in WO04/92360.

Viral epitopes/antigens include live, attenuated, split, and/or purifiedversions of any of the aforementioned.

Further sequences may also be derived from non-viral sources, forinstance, for instance, sequences encoding bacterial epitopes/antigens,including but not limited to, N. meningitides: a protein antigen from N.meningitides serogroup A, C, W135, Y, and/or B (1-7); an outer-membranevesicle (OMV) preparation from N. meningitides serogroup B. (8, 9, 10,11); a saccharide antigen, including LPS, from N. meningitides serogroupA, B, C W135 and/or Y, such as the oligosaccharide from serogroup C (seePCT/US99/09346; PCT IB98/01665; and PCT IB99/00103);

Streptococcus pneumoniae: a saccharide or protein antigen, particularlya saccharide from Streptococcus pneumoniae;

Streptococcus agalactiae: particularly, Group B streptococcus antigens;

Streptococcus pyogenes: particularly, Group A streptococcus antigens;

Enterococcus faecalis or Enterococcus faecium: Particularly atrisaccharide repeat or other Enterococcus derived antigens provided inU.S. Pat. No. 6,756,361;

Helicobacter pylori: including: Cag, Vac, Nap, HopX, HopY and/or ureaseantigen;

Bordetella pertussis: such as petussis holotoxin (PT) and filamentoushaemagglutinin (FHA) from B. pertussis, optionally also combination withpertactin and/or agglutinogens 2 and 3 antigen;

Staphylococcus aureus: including S. aureus type 5 and 8 capsularpolysaccharides optionally conjugated to nontoxic recombinantPseudomonas aeruginosa exotoxin A, such as StaphVAX™, or antigensderived from surface proteins, invasins (leukocidin, kinases,hyaluronidase), surface factors that inhibit phagocytic engulfment(capsule, Protein A), carotenoids, catalase production, Protein A,coagulase, clotting factor, and/or membrane-damaging toxins (optionallydetoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin,leukocidin);

Staphylococcus epidermis: particularly, S. epidermidis slime-associatedantigen (SAA);

Staphylococcus saprophyticus: (causing urinary tract infections)particularly the 160 kDa hemagglutinin of S. saprophyticus antigen;

Pseudomonas aeruginosa: particularly, endotoxin A, Wzz protein, P.aeruginosa LPS, more particularly LPS isolated from PAO1 (O5 serotype),and/or Outer Membrane Proteins, including Outer Membrane Proteins F(OprF) (Infect Immun. 2001 May; 69(5): 3510-3515);

Bacillus anthracis (anthrax): such as B. anthracis antigens (optionallydetoxified) from A-components (lethal factor (LF) and edema factor(EF)), both of which can share a common B-component known as protectiveantigen (PA);

Moraxella catarrhalis: (respiratory) including outer membrane proteinantigens (HMW-OMP), C-antigen, and/or LPS;

Yersinia pestis (plague): such as F1 capsular antigen (Infect Immun.2003 January; 71 (1)): 374-383, LPS (Infect Immun. 1999 October; 67(10):5395), Yersinia pestis V antigen (Infect Immun. 1997 November; 65(11):4476-4482);

Yersinia enterocolitica (gastrointestinal pathogen): particularly LPS(Infect Immun. 2002 August; 70(8): 4414);

Yersinia pseudotuberculosis: gastrointestinal pathogen antigens;

Mycobacterium tuberculosis: such as lipoproteins, LPS, BCG antigens, afusion protein of antigen 85B (Ag85B) and/or ESAT-6 optionallyformulated in cationic lipid vesicles (Infect Immun. 2004 October;72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenaseassociated antigens (Proc Natl Acad Sci USA. 2004 Aug. 24; 101(34):12652), and/or MPT51 antigens (Infect Immun. 2004 July; 72(7): 3829);

Legionella pneumophila (Legionnairs' Disease): L. pneumophilaantigens-optionally derived from cell lines with disrupted asd genes(Infect Immun. 1998 May; 66(5): 1898);

Rickettsia: including outer membrane proteins, including the outermembrane protein A and/or B (OmpB) (Biochim Biophys Acta. 2004 Nov. 1;1702(2):145), LPS, and surface protein antigen (SPA) (J Autoimmun. 1989June; 2 Suppl:81);

E. coli: including antigens from enterotoxigenic E. coli (ETEC),enteroaggregative E. coli (EAggEC), diffulsely adhering E. coli (DAEC),enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E. coli(EHEC);

Vibrio cholerae: including proteinase antigens, LPS, particularlylipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specificpolysaccharides, V. cholera 0139, antigens of IEM108 vaccine (InfectImmun. 2003 October; 71(10):5498-504), and/or Zonula occludens toxin(Zot);

Salmonella typhi (typhoid fever): including capsular polysaccharidespreferably conjugates (Vi, i.e. vax-TyVi);

Salmonella typhimurium (gastroenteritis): antigens derived therefrom arecontemplated for microbial and cancer therapies, including angiogenesisinhibition and modulation of flk;

Listeria monocytogenes (systemic infections in immunocompromised orelderly people, infections of fetus): antigens derived from L.monocytogenes are preferably used as carriers/vectors forintracytoplasmic delivery of conjugates/associated compositions of thepresent invention;

Porphyromonas gingivalis: particularly, P. gingivalis outer membraneprotein (OMP);

Tetanus: such as tetanus toxoid (TT) antigens, preferably used as acarrier protein in conjunction/conjugated with the compositions of thepresent invention;

Diphtheria: such as a diphtheria toxoid, preferably CRM₁₉₇, additionallyantigens capable of modulating, inhibiting or associated with ADPribosylation are contemplated forcombination/co-administration/conjugation with the compositions of thepresent invention, the diphtheria toxoids are preferably used as carrierproteins;

Borrelia burgdorferi (Lyme disease): such as antigens associated withP39 and P13 (an integral membrane protein, Infect Immun. 2001 May;69(5): 3323-3334), VlsE Antigenic Variation Protein (J Clin Microbiol.1999 December; 37(12): 3997);

Haemophilus influenzae B: such as a saccharide antigen therefrom;

Klebsiella: such as an OMP, including OMP A, or a polysaccharideoptionally conjugated to tetanus toxoid;

Neiserria gonorrhoeae: including, a Por (or porin) protein, such as PorB(see Zhu et al., Vaccine (2004) 22:660-669), a transferring bindingprotein, such as ThpA and TbpB (See Price et al., Infection and Immunity(2004) 71(1):277-283), a opacity protein (such as Opa), areduction-modifiable protein (Rmp), and outer membrane vesicle (OMV)preparations (see Plante et al., J Infectious Disease (2000)182:848-855), also see e.g. WO99/24578, WO99/36544, WO99/57280,WO021079243);

Chlamydia pneumoniae: particularly C. pneumoniae protein antigens;

Chlamydia trachomatis: including antigens derived from serotypes A, B,Ba and C are (agents of trachoma, a cause of blindness), serotypes L₁,L₂ & L₃ (associated with Lymphogranuloma venereum), and serotypes, D-K;

Treponema pallidum (Syphilis): particularly a TmpA antigen; and

Haemophilus ducreyi (causing chancroid): including outer membraneprotein (DsrA).

Where not specifically referenced, further bacterial antigens may becapsular antigens, polysaccharide antigens or protein antigens of any ofthe above. Further bacterial antigens may also include an outer membranevesicle (OMV) preparation. Additionally, antigens include live,attenuated, split, and/or purified versions of any of the aforementionedbacteria. The bacterial or microbial derived antigens may begram-negative or gram-positive and aerobic or anaerobic. Additionally,any of the above bacterial-derived saccharides (polysaccharides, LPS,LOS or oligosaccharides) can be conjugated to another agent or antigen,such as a carrier protein (for example CRM₁₉₇). Such conjugation may bedirect conjugation effected by reductive amination of carbonyl moietieson the saccharide to amino groups on the protein, as provided in U.S.Pat. No. 5,360,897 and Can J Biochem Cell Biol. 1984 May; 62(5):270-5.Alternatively, the saccharides can be conjugated through a linker, suchas, with succinamide or other linkages provided in BioconjugateTechniques, 1996 and CRC, Chemistry of Protein Conjugation andCross-Linking, 1993

Further sequences may also be those encoding fungal antigens, includingthose described in: U.S. Pat. Nos. 4,229,434 and 4,368,191 forprophylaxis and treatment of trichopytosis caused by Trichophytonmentagrophytes; U.S. Pat. Nos. 5,277,904 and 5,284,652 for a broadspectrum dermatophyte vaccine for the prophylaxis of dermatophyteinfection in animals, such as guinea pigs, cats, rabbits, horses andlambs, these antigens comprises a suspension of killed T. equinum, T.mentagrophytes (var. granulare), M. canis and/or M. gypseum in aneffective amount optionally combined with an adjuvant; U.S. Pat. Nos.5,453,273 and 6,132,733 for a ringworm vaccine comprising an effectiveamount of a homogenized, formaldehyde-killed fingi, i.e., Microsporumcanis culture in a carrier; U.S. Pat. No. 5,948,413 involvingextracellular and intracellular proteins for pythiosis. Additionalantigens identified within antifingal vaccines include Ringvac bovisLTF-130 and Bioveta.

Further, fungal antigens for use herein may be derived fromDermatophytres, including: Epidermophyton floccusum, Microsporumaudouini, Microsporum canis, Microsporum distortum, Microsporum equinum,Microsporum gypsum, Microsporum nanum, Trichophyton concentricum,Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum,Trichophyton megnini, Trichophyton mentagrophytes, Trichophytonquinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophytontonsurans, Trichophyton verrucosum, T. verrucosum var. album, var.discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophytonfaviforme.

Fungal pathogens for use as antigens or in derivation of antigens inconjunction with the compositions of the present invention compriseAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi,Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata,Candida krusei, Candida parapsilosis, Candida stellatoidea, Candidakusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis,Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum,Histoplasma capsulatum, Klebsiella pneumoniae, Paracoccidioidesbrasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporumovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomycespombe, Scedosporium apiosperum, Sporothrix schencki, Trichosporonbeigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp.,Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp.,Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierellaspp, Cunninghamella spp, and Saksenaea spp.

Other fungi from which antigens are derived include Alternaria spp,Curvularia spp, Helminthosporium spp, Fusariuin spp, Aspergillus spp,Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp,Pithomyces spp, and Cladosporium spp.

Processes for producing a fungal antigens are well known in the art (seeU.S. Pat. No. 6,333,164). In a preferred method a solubilized fractionextracted and separated from an insoluble fraction obtainable fromfungal cells of which cell wall has been substantially removed or atleast partially removed, characterized in that the process comprises thesteps of: obtaining living fungal cells; obtaining fungal cells of whichcell wall has been substantially removed or at least partially removed;bursting the fungal cells of which cell wall has been substantiallyremoved or at least partially removed; obtaining an insoluble fraction;and extracting and separating a solubilized fraction from the insolublefraction.

Further sequences (e.g., transgenes) also include, but are not limitedto, sequences encoding tumor antigens/epitopes. Tumor antigens can be,for example, peptide-containing tumor antigens, such as a polypeptidetumor antigen or glycoprotein tumor antigens. A tumor antigen can alsobe, for example, a saccharide-containing tumor antigen, such as aglycolipid tumor antigen or a ganglioside tumor antigen. The tumorantigen can further be, for example, a polynucleotide-containing tumorantigen that expresses a polypeptide-containing tumor antigen, forinstance, an RNA vector construct or a DNA vector construct, such asplasmid DNA. Tumor antigens encompass a wide variety of molecules, suchas (a) polypeptide-containing tumor antigens, including polypeptides(which can range, for example, from 8-20 amino acids in length, althoughlengths outside this range are also common), lipopolypeptides andglycoproteins, (b) saccharide-containing tumor antigens, includingpoly-saccharides, mucins, gangliosides, glycolipids and glycoproteins,and (c) polynucleotides that express antigenic polypeptides.

The tumor antigens can be, for example, (a) full length moleculesassociated with cancer cells, (b) homologs and modified forms of thesame, including molecules with deleted, added and/or substitutedportions, and (c) fragments of the same. Tumor antigens can be providedin recombinant form. Tumor antigens include, for example, classI-restricted antigens recognized by CD8+ lymphocytes or classII-restricted antigens recognized by CD4+ lymphocytes.

Numerous tumor antigens are known in the art, including: (a)cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE,BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2,MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which canbe used, for example, to address melanoma, lung, head and neck, NSCLC,breast, gastrointestinal, and bladder tumors), (b) mutated antigens, forexample, p53 (associated with various solid tumors, e.g., colorectal,lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma,pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g.,melanoma), MUM1 (associated with, e.g., melanoma), caspase-8 (associatedwith, e.g., head and neck cancer), CIA 0205 (associated with, e.g.,bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g.,melanoma), TCR (associated with, e.g., T-cell non-Hodgkins lymphoma),BCR-ab1 (associated with, e.g., chronic myelogenous leukemia),triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-FUT, (c)over-expressed antigens, for example, Galectin 4 (associated with, e.g.,colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin'sdisease), proteinase 3 (associated with, e.g., chronic myelogenousleukemia), WT 1 (associated with, e.g., various leukemias), carbonicanhydrase (associated with, e.g., renal cancer), aldolase A (associatedwith, e.g., lung cancer), PRAME (associated with, e.g., melanoma),HER-2/neu (associated with, e.g., breast, colon, lung and ovariancancer), alpha-fetoprotein (associated with, e.g., hepatoma), KSA(associated with, e.g., colorectal cancer), gastrin (associated with,e.g., pancreatic and gastric cancer), telomerase catalytic protein,MUC-1 (associated with, e.g., breast and ovarian cancer), G-250(associated with, e.g., renal cell carcinoma), p53 (associated with,e.g., breast, colon cancer), and carcinoembryonic antigen (associatedwith, e.g., breast cancer, lung cancer, and cancers of thegastrointestinal tract such as colorectal cancer), (d) shared antigens,for example, melanoma-melanocyte differentiation antigens such asMART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor,tyrosinase, tyrosinase related protein-1/TRP1 and tyrosinase relatedprotein-2/TRP2 (associated with, e.g., melanoma), (e) prostateassociated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2,associated with e.g., prostate cancer, (f) immunoglobulin idiotypes(associated with myeloma and B cell lymphomas, for example), and (g)other tumor antigens, such as polypeptide- and saccharide-containingantigens including (i) glycoproteins such as sialyl Tn and sialyl Le^(x)(associated with, e.g., breast and colorectal cancer) as well as variousmucins; glycoproteins may be coupled to a carrier protein (e.g., MUC-1may be coupled to KLH); (ii) lipopolypeptides (e.g., MUC-1 linked to alipid moiety); (iii) polysaccharides (e.g., Globo H synthetichexasaccharide), which may be coupled to a carrier proteins (e.g., toKLH), (iv) gangliosides such as GM2, GM12, GD2, GD3 (associated with,e.g., brain, lung cancer, melanoma), which also may be coupled tocarrier proteins (e.g., KLH).

Additional tumor antigens which are known in the art include p15,Horn/MeI-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barrvirus antigens, EBNA, human papillomavirus (HPV) antigens, including E6and E7, hepatitis B and C virus antigens, human T-cell lymphotropicvirus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1,TAG-72-4, CA 19-9, CA 724, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7,43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associatedprotein), TAAL6, TAG72, TLP, TPS, and the like. These as well as othercellular components are described for example in United States PatentApplication 20020007173 and references cited therein.

Polynucleotide-containing antigens in accordance with the presentinvention typically comprise polynucleotides that encode polypeptidecancer antigens such as those listed above. Preferredpolynucleotide-containing antigens include DNA or RNA vector constructs,such as plasmid vectors (e.g., pCMV), which are capable of expressingpolypeptide cancer antigens in vivo.

Tumor antigens may be derived, for example, from mutated or alteredcellular components. After alteration, the cellular components no longerperform their regulatory functions, and hence the cell may experienceuncontrolled growth. Representative examples of altered cellularcomponents include ras, p53, Rb, altered protein encoded by the Wilms'tumor gene, ubiquitin, mucin, protein encoded by the DCC, APC, and MCCgenes, as well as receptors or receptor-like structures such as neu,thyroid hormone receptor, platelet derived growth factor (PDGF)receptor, insulin receptor, epidermal growth factor (EGF) receptor, andthe colony stimulating factor (CSF) receptor. These as well as othercellular components are described for example in U.S. Pat. No. 5,693,522and references cited therein.

Additional information on cancer or tumor antigens can be found, forexample, in Moingeon P, “Cancer vaccines,” Vaccine, 2001, 19:1305-1326;Rosenberg S A, “Progress in human tumor immunology and immunotherapy,”Nature, 2001, 411:380-384; Dermine, S. et al, “Cancer Vaccines andImmunotherapy,” British Medical Bulletin, 2002, 62, 149-162;Espinoza-Delgado I., “Cancer Vaccines,” The Oncologist, 2002, 7(suppl3):20-33; Davis, I. D. et al., “Rational approaches to humancancer immunotherapy,” Journal of Leukocyte Biology, 2003, 23: 3-29; Vanden Eynde B, et al., “New tumor antigens recognized by T cells,” Curr.Opin. Immunol., 1995, 7:674-81; Rosenberg S A, “Cancer vaccines based onthe identification of genes encoding cancer regression antigens,Immunol. Today, 1997, 18:175-82; Offringa R et al., “Design andevaluation of antigen-specific vaccination strategies against cancer,”Current Opin. Immunol., 2000, 2:576-582; Rosenberg S A, “A new era forcancer immunotherapy based on the genes that encode cancer antigens,”Immunity, 1999, 10:281-7; Sahin U et al., “Serological identification ofhuman tumor antigens,” Curr. Opin. Immunol., 1997, 9:709-16; Old L J etal., “New paths in human cancer serology,” J. Exp. Med., 1998,187:1163-7; Chaux P, et al., “Identification of MAGE-3 epitopespresented by HLA-DR molecules to CD4(+) T lymphocytes,” J. Exp. Med.,1999, 189:767-78; Gold P, et al., “Specific carcinoembryonic antigens ofthe human digestive system,” J. Exp. Med., 1965, 122:467-8; Livingston PO, et al., Carbohydrate vaccines that induce antibodies against cancer:Rationale,” Cancer Immunol. Immunother., 1997, 45:1-6; Livingston P O,et al., Carbohydrate vaccines that induce antibodies against cancer:Previous experience and future plans,” Cancer Immunol. Immunother.,1997, 45:10-9; Taylor-Papadimitriou J, “Biology, biochemistry andimmunology of carcinoma-associated mucins,” Immunol. Today, 1997,18:105-7; Zhao X-J et al., “GD2 oligosaccharide: target for cytotoxic Tlymphocytes,” J. Exp. Med., 1995, 182:67-74; Theobald M, et al.,“Targeting p53 as a general tumor antigen,” Proc. Natl. Acad. Sci. USA,1995, 92:11993-7; Gaudernack G, “T cell responses against mutant ras: abasis for novel cancer vaccines,” Immunotechnology, 1996, 2:3-9; WO91/02062; U.S. Pat. No. 6,015,567; WO 01/08636; WO 96/30514; U.S. Pat.No. 5,846,538; and U.S. Pat. No. 5,869,445.

Further sequences also include sequences encoding cytokines suchinterleukin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3),interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocytemacrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha(IL-1), interleukin-11 (IL-11), MIP-1, tumor necrosis factor (TNF),leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO) andflt3 ligand, commercially available from several vendors such as, forexample, Genzyme (Framingham, Mass.), Genentech (South San Francisco,Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems and Immunex(Seattle, Wash.). Additional sequences are described below. Also,variations on the orientation of the Gag and other coding sequences,relative to each other, are described below.

Although exemplified with regard to subtype B isolate SF2, it will beapparent that sequences as described herein can readily be derived fromother HIV isolates, see, e.g., Myers et al. Los Alamos Database, LosAlamos National Laboratory, Los Alamos, N. Mex. (1992); Myers et al.,Human Retroviruses and Aids, 1997, Los Alamos, N. Mex. Los AlamosNational Laboratory. Synthetic expression cassettes can be generatedusing such coding sequences, as starting material by following theteachings of the present specification.

5. Expression Of Synthetic Sequences

The synthetic sequences encoding HIV polypeptides described hereinincrease expression and secretion of HIV polypeptides. In particular,sequences including Gag, pol, tat, rev and nef expressed as apolyprotein are disclosed. Many of these sequences exhibit increasedexpression and secretion as compared to sequences encoding wild-type.Data shown below also indicates that when expressed as a polyprotein,the sequences may exhibit increased expression of polypeptides ascompared to expression obtained from modified (synthetic) Gag- andpoll-encoding sequences. Furthermore, constructs as described herein inwhich sequences encoding Tat, rev and/or nef are 5′ relative tosequences encoding Gag and/or Pol may exhibit increased expression andsecretion as compared to constructs having the reverse orientation(i.e., Tat, rev and/or nef are 3′ relative to sequences encoding Gagand/or Pol).

Preferably, the multiple sequences are cloned in-frame into a singlevector such that multiple polynucleotides encoding a more than one geneproduct (or portion thereof) (e.g., polycistronic coding sequences) toproduce a single polyprotein. Optionally, the polyprotein may proteincleavage sites between one or more of the polypeptides comprising thepolyprotein.

The present invention also includes co-transfection with multiple,monocistronic expression cassettes, as well as, co-transfection with oneor more multi-cistronic expression cassettes, or combinations thereof.For example, a bicistronic construct may be made where the codingsequences for the different HIV polypeptides are under the control of asingle CMV promoter and, between the two coding sequences, an IRES(internal ribosome entry site EMCV IRES); Kozak, M., Critical Reviews inBiochemistry and Molecular Biology 27(45):385-402, 1992; Witherell, G.W., et al., Virology 214:660-663, 1995) sequence is introduced after thefirst HIV coding sequence and before the second HIV coding sequence.

To evaluate expression levels and/or secretion, sequences describedherein can be cloned into a number of different vectors including butnot limited to, prokaryotic vectors and eukaryotic expression vectors,including, a transient expression vector, CMV-promoter-based mammalianvectors, and a shuttle vector for use in baculovirus expression systems.

Insect cell expression systems, such as baculovirus systems, are knownto those of skill in the art and described in, e.g., Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987).Materials and methods for baculovirus/insert cell expression systems arecommercially available in kit form from, inter alia, Invitrogen, SanDiego Calif. Similarly, bacterial and mammalian cell expression systemsare also known in the art and described in, e.g., Yeast GeneticEngineering (Barr et al., eds., 1989) Butterworths, London.

These vectors can then be transfected into a several different celltypes, including a variety of mammalian cell lines (293, RD, COS-7, andCHO, cell lines available, for example, from the A.T.C.C.). The celllines are then cultured under appropriate conditions and the levels ofany appropriate polypeptide product can be evaluated in supernatants.For example, p24 or p55 can be used to determine Gag expression.Further, modified polypeptides can also be used, for example, other Gagpolypeptides. The results of these assays demonstrate that expression ofsynthetic HIV polypeptide-encoding sequences are significantly higherthan corresponding wild-type sequences.

Further, Western Blot analysis can be used to show that cells comprisingthe synthetic polynucleotides (e.g., expression cassettes comprisingthese polynucleotides) produce the expected protein at higher per-cellconcentrations than cells containing the native sequences. Proteinanalysis can be conducted using any suitable assay, for example theOdyssey™ Infrared Antibody Detection Systems (Li-Cor Biosciences,Lincoln, Nebr.). The HIV proteins can be seen in both cell lysates andsupernatants. (Example 3, FIGS. 3 and 4).

Fractionation of the supernatants from mammalian cells transfected asdescribed herein can be used to show that vector; comprising thesynthetic sequences described herein provide superior production of HIVproteins.

Efficient expression of these HIV-containing polypeptides in mammaliancell lines provides the following benefits: increased secretion may leadto increased uptake of proteins by antigen presenting cells, which inturn may lead to increased immunogenicity, for example when delivered(e.g., subcutaneously, intradermally, or mucosally) to APC-richenvironment; increased expression may lead to increased primingefficiency and/or increased cross-priming efficiency (where cellsadjacent to those which take up the DNA are also primed), for example incombination with alphavirus replicons; the polypeptides are free ofbaculovirus contaminants; production by established methods approved bythe FDA; increased purity; greater yields (relative to native codingsequences); and a novel method of producing the HIV-containingpolypeptides in CHO cells which is not feasible in the absence of theincreased expression (as compared, for example, to native sequences)obtained using the constructs of the present invention.

Mammalian cell lines are known in the art and include immortalized celllines available from the American Type Culture Collection (ATCC), suchas, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells,baby hamster kidney (BHK) cells, monkey kidney cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, PERC.6 cells which aredescribed, for example, in WO 01/38362 and WO 02/40665, incorporated byreference herein in their entireties, as well as deposited under ECACCdeposit number 96022940), as well as others.

Mammalian sources of cells include, but are not limited to, human ornon-human primate (e.g., MRC-5 (ATCC CCL-1711), WI-38 (ATCC CCL-75),fetal rhesus lung cells (ATCC CL-160), human embryonic kidney cells (293cells, typically transformed by sheared adenovirus type 5 DNA), VEROcells from monkey kidneys), horse, cow (e.g., MDBK cells), sheep, dog(e.g., MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK33016, deposit number DSM ACC 2219 as described in WO 97/37001), cat,and rodent (e.g., hamster cells such as BHK21-F, HKCC cells, or Chinesehamster ovary cells (CHO cells)), and may be obtained from a widevariety of developmental stages, including for example, adult, neonatal,fetal, and embryo.

Other exemplary Mammalian cell lines include, but are not limited to,HT1080, RD, COS-7, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT4,H9, PM1, CEM, and CEMX174, such cell lines are available, for example,from the A.T.C.C.). Insect cells for use with baculovirus expressionvectors include, inter alia, Aedes aegypti, Autographa californica,Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, andTrichoplusia ni.

Further, synthetic sequences of the present invention can also beintroduced into yeast vectors which, in turn, can be transformed intoand efficiently expressed by yeast cells (Saccharomyces cerevisea; usingvectors as described in Rosenberg, S. and Tekamp-Olson, P., U.S. PatentNo. RE35,749, issued, Mar. 17, 1998, herein incorporated by reference).Yeast cells include, inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenual polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica.

In addition to the mammalian and insect vectors, the syntheticpolynucleotides of the present invention can also be produced(expressed) using an avian expression system. Avian cell expressionsystems are known in the art and described in, e.g., U.S. Pat. Nos.5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668; EuropeanPatent No. EP 0787180B; WO 03/043415; and WO 03/076601. Avian cell linesare known in the art and include embryonic germ cell lines; andembryonic stem (ES) cell lines. Avian sources of cells include, but arenot limited to, chicken ES cells (EBx® cell lines), chicken EG cells,turkey ES or EG cells, quail ES or EG cells, pheasant ES or EG cells.

In addition to the mammalian, avian and insect vectors, the syntheticpolynucleotides of the present invention can be incorporated into avariety of expression vectors using selected expression controlelements. Appropriate vectors and control elements for any given celltype can be selected by one having ordinary skill in the art in view ofthe teachings of the present specification and information known in theart.

For example, a suitable vector may include control elements operablylinked to the desired coding sequence, which allow for the expression ofthe gene in a selected cell-type. For example, typical promoters formammalian cell expression include the SV40 early promoter, a CMVpromoter such as the CMV immediate early promoter (a CMV promoter caninclude intron A), RSV, HIV-Ltr, the mouse mammary tumor virus LTRpromoter (MMLV-ltr), the adenovirus major late promoter (Ad MLP), andthe herpes simplex virus promoter, among others. Other nonviralpromoters, such as a promoter derived from the murine metallothioneingene, will also find use for mammalian expression. Typically,transcription termination and polyadenylation sequences will also bepresent, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook, et al., supra, as well as a bovine growth hormoneterminator sequence. Introns, containing splice donor and acceptorsites, may also be designed into the constructs for use with the presentinvention (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986).

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence (Chapman et al., Nuc. Acids Res.(1991) 19:3979-3986).

The desired synthetic polypeptide encoding sequences can be cloned intoany number of commercially available vectors to generate expression ofthe polypeptide in an appropriate host system. These systems include,but are not limited to, the following: baculovirus expression {Reilly,P. R., et al., Baculovirus Expression Vectors: A Laboratory Manual(1992); Beames, et al., Biotechniques 11:378 (1991); Pharmingen;Clontech, Palo Alto, Calif.)}, vaccinia expression {Earl, P. L., et al.,“Expression of proteins in mammalian cells using vaccinia” In CurrentProtocols in Molecular Biology (F. M. Ausubel, et al. Eds.), GreenePublishing Associates & Wiley Interscience, New York (1991); Moss, B.,et al., U.S. Pat. No. 5,135,855, issued 4 Aug. 1992}, expression inbacteria {Ausubel, F. M., et al., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., Media Pa.; Clontech}, expression inyeast {Rosenberg, S, and Tekamp-Olson, P., U.S. Pat. No. RE35,749,issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R.,U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated byreference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93 (1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992);Goeddel, D. V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R.Fink, Methods in Enzymology 194 (1991)}, expression in mammalian cells{Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary(CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983);1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman,R. J., “Selection and coamplification of heterologous genes in mammaliancells,” in Methods in Enzmology, vol. 185, pp 537-566. Academic Press,Inc., San Diego Calif. (1991)}, and expression in plant cells {plantcloning vectors, Clontech Laboratories, Inc., Palo Alto, Calif., andPharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al.,J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol.Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in PlantMolecular Biology Manual A3:1-19 (1988); Miki, B. L. A., et al., pp.249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al.,eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology:Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley,1997; Miglani, Gurbachan Dictionary of Plant Genetics and MolecularBiology, New York, Food Products Press, 1998; Henry, R. J., PracticalApplications of Plant Molecular Biology, New York, Chapman & Hall,1997}.

As noted above, the expression vectors typically contain codingsequences and expression control elements that allow expression of thecoding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector. Translational control elementshave been reviewed by M. Kozak (e.g., Kozak, M., Mamm. Genome7(8):563-574, 1996; Kozak, M., Biochimie 76(9):815-821, 1994; Kozak, M.,J Cell Biol 108(2):229-241, 1989; Kozak, M., and Shatkin, A. J., MethodsEnzymol 60:360-375, 1979).

Expression in yeast systems has the advantage of commercial production.Recombinant protein production by vaccinia and CHO cell line has theadvantage of being mammalian expression systems. Further, vaccinia virusexpression has several advantages including the following: (i) its widehost range; (ii) faithful post-transcriptional modification, processing,folding, transport, secretion, and assembly of recombinant proteins;(iii) high level expression of relatively soluble recombinant proteins;and (iv) a large capacity to accommodate foreign DNA.

The recombinantly expressed polypeptides from synthetic HIVpolypeptide-encoding sequences are typically isolated from lysed cellsor culture media. Purification can be carried out by methods known inthe art including salt fractionation, ion exchange chromatography, gelfiltration, size-exclusion chromatography, size-fractionation, andaffinity chromatography. Immunoaffinity chromatography can be employedusing antibodies generated based on, for example, HIV antigens.

Advantages of expressing the proteins of the present invention usingmammalian cells include, but are not limited to, the following:well-established protocols for scale-up production; the ability toproduce neutralizing antibodies; cell lines are suitable to meet goodmanufacturing process (GMP) standards; culture conditions for mammaliancells are known in the art.

Synthetic HIV 1 polynucleotides are described herein, see, for example,the figures. Various forms of the different embodiments of theinvention, described herein, may be combined.

6. DNA Immunization And Gene Delivery

DNA immunization using synthetic sequences of the present invention canbe performed, for example, as follows. Mice are immunized with anexpression cassette comprising, proceeding in a 5′ to 3′ direction,synthetic tat-, rev- and/or nef-encoding sequences followed by syntheticGag- and/or Pol-encoding sequences. Other mice are immunized with atat/rev/nef/Gag/Pol wild type expression cassettes and/or cassettes inwhich synthetic Gag- and/or Pol-encoding sequences are 5′ to synthetictat-, rev- and/or nef-encoding sequences. Further, animals arepreferably immunized with expression cassettes comprising wild typesequences as well as other synthetic sequences (e.g., Gag, Pol, GagPol,TatRevNef, etc.).

Other HIV antigens of particular interest to be used in the practice ofthe present invention include, but are not limited to, Env, vif, vpu,vpr, and other HIV antigens or epitopes derived therefrom. Theseantigens may be synthetic (as described herein) or wild-type. Further,the packaging cell line may contain only nef, and HIV-1 (also known asHTLV-III, LAV, ARV, etc.), including, but not limited to, antigens (bothnative and modified) from a variety of isolates including, but notlimited to, HIVIIIb, HIVSF2, HIV-ISF162, HIV-ISF170, HIV_(LAV), HIVLAI,HIVMN, HIV-1CM235, HIV-1US4, other HIV-1 strains from diverse subtypes(e.g., subtypes, A through K, N and O), HIV-2 strains and diversesubtypes (e.g., HIV-2UC1 and HIV-2UC2). See, e.g., Myers, et al., LosAlamos Database, Los Alamos National Laboratory, Los Alamos, N. Mex.;Myers, et al., Human Retroviruses and Aids, 1990, Los Alamos, N. Mex.:Los Alamos National Laboratory.

To evaluate efficacy, DNA immunization can be performed, for instance asdescribed in Example 4. Animals (e.g., mice, guinea pigs, rabbits ornon-human primates) are immunized with the poll istronic syntheticsequences (e.g., expression cassettes) having sequences in various; 5′to 3′ orders, unicistronic synthetic sequences (e.g., Gag or Pol),bicistronic sequences (e.g., GagPol) and the wild type Env sequences.Immunizations with the polynucleotides will show that the syntheticsequences provide a clear improvement of immunogenicity relative to thenative sequences. Also, the second boost immunization will induce asecondary immune response, for example, after approximately two weeks.

6.1 Delivery

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. Furthermore, the desiredgene can be isolated directly from cells and tissues containing thesame, using standard techniques, such as phenol extraction and PCR ofcDNA or genomic DNA. See, e.g., Sambrook et al., supra, for adescription of techniques used to obtain and isolate DNA. The gene ofinterest can also be produced synthetically, rather than cloned. Thenucleotide sequence can be designed with the appropriate codons for theparticular amino acid sequence desired. In general, one will selectpreferred codons for the intended host in which the sequence will beexpressed. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature (1981) 292:756;Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem.(1984) 259:6311; Stemmer, W.P.C., (1995) Gene 164:49-53.

Next, the gene sequence encoding the desired antigen can be insertedinto a vector. The vector can also include control elements operablylinked to the coding sequence, which allow for the expression of thegene in vivo in the subject species. For example, typical promoters formammalian cell expression include the SV40 early promoter, a CMVpromoter such as the CMV immediate early promoter, the mouse mammarytumor virus LTR promoter, the adenovirus major late promoter (Ad MLP),and the herpes simplex virus promoter, among others. Other nonviralpromoters, such as a promoter derived from the murine metallothioneingene, will also find use for mammalian expression. Typically,transcription termination and polyadenylation sequences will also bepresent, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Furthermore, plasmids can be constructed which include a chimericantigen-coding gene sequences, encoding, e.g., multipleantigens/epitopes of interest, for example derived from more than oneviral isolate.

The antigen coding sequences precede or follow the synthetic codingsequence and the chimeric transcription unit an may have a single openreading frame encoding both the antigen of interest and the syntheticcoding sequences. Alternatively, polycistronic cassettes can beconstructed allowing expression of multiple antigens from a single mRNAusing the EMCV IRES, or the like.

Once complete, the constructs are used for nucleic acid immunizationusing standard gene delivery protocols. Methods for gene delivery areknown in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,5,589,466. Genes can be delivered either directly to the vertebratesubject or, alternatively, delivered ex vivo, to cells derived from thesubject and the cells reimplanted in the subject.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering the polynucleotides of thepresent invention is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Additional viral vectors that will find use for delivering the nucleicacid molecules encoding the antigens of interest include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing thegenes can be constructed as follows. The DNA encoding the particularsynthetic HIV polypeptide coding sequence is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells that aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the codingsequences of interest into the viral genome. The resultingTK-recombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the genes. Recombinant avipox viruses,expressing immunogens from mammalian pathogens, are known to conferprotective immunity when administered to non-avian species. The use ofan avipox vector is particularly desirable in human and other mammalianspecies since members of the avipox genus can only productivelyreplicate in susceptible avian species and therefore are not infectivein mammalian cells. Methods for producing recombinant avipoxviruses areknown in the art and employ genetic recombination, as described abovewith respect to the production of vaccinia viruses. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, will also find use as viral vectors for deliveringthe polynucleotides of the present invention (for example, a syntheticEnv-polypeptide encoding expression cassette). For a description ofSindbis-virus derived vectors useful for the practice of the instantmethods, see, Dubensky et al., J. Virol. (1996) 70:508-519; andInternational Publication Nos. WO 95/07995 and WO 96/17072; as well as,Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1,1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4,1998, both herein incorporated by reference.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the coding sequencesof interest in a host cell. In this system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAthat is then translated into protein by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase that in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

Synthetic sequences of interest can also be delivered without a viralvector. For example, the synthetic sequences (or expression cassettes)can be packaged in liposomes prior to delivery to the subject or tocells derived therefrom. Lipid encapsulation is generally accomplishedusing liposomes that are able to stably bind or entrap and retainnucleic acid. The ratio of condensed DNA to lipid preparation can varybut will generally be around 1:1 (mg DNA:micromoles lipid), or more oflipid. For a review of the use of liposomes as carriers for delivery ofnucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzyrnology (1983), Vol.101, pp. 512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA, (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081);and purified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198;PCT Publication No. WO 90/11092 for a description of the synthesis ofDOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles.(MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA(1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta(1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acacl. Sci. USA(1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA(1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szokaand Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; andSchaefer-Ridder et al., Science (1982) 215:166.

The DNA and/or protein antigen(s) can also be delivered in cochleatelipid compositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The synthetic sequences (and/or expression cassettes) of interest mayalso be encapsulated, adsorbed to, or associated with, particulatecarriers. Such carriers present multiple copies of a selected antigen tothe immune system and promote trapping and retention of antigens inlocal lymph nodes. The particles can be phagocytosed by macrophages andcan enhance antigen presentation through cytokine release. Examples ofparticulate carriers include those derived from polymethyl methacrylatepolymers, as well as microparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,Pharm. Res. (1993) 10:362-368; McGee J P, et al., J. Microencapsul.14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993.Suitable microparticles may also be manufactured in the presence ofcharged detergents, such as anionic or cationic detergents, to yieldmicroparticles with a surface having a net negative or a net positivecharge. For example, microparticles manufactured with anionicdetergents, such as hexadecyltrimethylammonium bromide (CTAB), i.e.CTAB-PLG microparticles, adsorb negatively charged macromolecules, suchas DNA. (see, e.g., Int'l Application Number PCT/US99/17308).

Furthermore, other particulate systems and polymers can be used for thein vivo or ex vivo delivery of the gene of interest. For example,polymers such as polylysine, polyarginine, polyomithine, spermine,spermidine, as well as conjugates of these molecules, are useful fortransferring a nucleic acid of interest. Similarly, DEAEdextran-mediated transfection, calcium phosphate precipitation orprecipitation using other insoluble inorganic salts, such as strontiumphosphate, aluminum silicates including bentonite and kaolin, chromicoxide, magnesium silicate, talc, and the like, will find use with thepresent methods. See, e.g., Feigner, P. L., Advanced Drug DeliveryReviews (1990) 5:163-187, for a review of delivery systems useful forgene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov. 3, 1998, herein incorporated by reference) mayalso be used for delivery of a construct of the present invention.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for deliveringsynthetic sequences and vectors of the present invention. The particlesare coated with the synthetic sequences (and/or expression cassette(s))to be delivered and accelerated to high velocity, generally under areduced atmosphere, using a gun powder discharge from a “gene gun.” Fora description of such techniques, and apparatuses useful therefore, see,e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022;5,371,015; and 5,478,744. Also, needle-less injection systems can beused (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc.,Portland, Oreg.). Direct delivery of compositions comprising thesynthetic sequences described herein in vivo will generally beaccomplished with or without viral vectors, as described above, byinjection using either a conventional syringe or a gene gun, such as theAccell® gene delivery system (PowderJect Technologies, Inc., Oxford,England).

The constructs can be injected either subcutaneously, epidermally,intradermally, intramucosally such as nasally, rectally and vaginally,intraperitoneally, intravenously, orally or intramuscularly. Delivery ofDNA into cells of the epidermis is particularly preferred as this modeof administration provides access to skin-associated lymphoid cells andprovides for a transient presence of DNA in the recipient. Other modesof administration include oral and pulmonary administration,suppositories, needle-less injection, transcutaneous and transdermalapplications.

The sequences described herein may also be administered using in vivoelectroporation techniques. Efficient in vivo expression of plasmidencoded genes by electrical permeabilization (electroporation) has beendescribed (see, e.g., Zucchelli et al. (2000) J. Virol. 74:11598-11607;Banga et al. (1998) Trends Biotechnol. 10:408-412; Heller et al. (1996)Febs Lett. 389:225-228; Mathiesen et al. (1999) Gene Ther. 4:508-514;Mir et al. (1999) Proc. Nat'l Acad. Sci. USA 8:4262-4267; Nishi et al.(1996) Cancer Res. 5:1050-1055).

6.2 Ex Vivo Delivery

In one embodiment, T cells, and related cell types (including but notlimited to antigen presenting cells, such as, macrophage, monocytes,lymphoid cells, dendritic cells, B-cells, T-cells, stem cells, andprogenitor cells thereof), can be used for ex vivo delivery of thesynthetic sequences of the present invention. T cells can be isolatedfrom peripheral blood lymphocytes (PBLs) by a variety of proceduresknown to those skilled in the art. For example, T cell populations canbe “enriched” from a population of PBLs through the removal of accessoryand B cells. In particular, T cell enrichment can be accomplished by theelimination of non-T cells using anti-MHC class II monoclonalantibodies. Similarly, other antibodies can be used to deplete specificpopulations of non-T cells. For example, anti-Ig antibody molecules canbe used to deplete B cells and anti-MacI antibody molecules can be usedto deplete macrophages.

T cells can be further fractionated into a number of differentsubpopulations by techniques known to those skilled in the art. Twomajor subpopulations can be isolated based on their differentialexpression of the cell surface markers CD4 and CD8. For example,following the enrichment of T cells as described above, CD4+ cells canbe enriched using antibodies specific for CD4 (see Coligan et al.,supra). The antibodies may be coupled to a solid support such asmagnetic beads. Conversely, CD8+ cells can be enriched through the useof antibodies specific for CD4 (to remove CD4+ cells), or can beisolated by the use of CD8 antibodies coupled to a solid support. CD4lymphocytes from HIV-1 infected patients can be expanded ex vivo, beforeor after transduction as described by Wilson et. al. (1995) J. Infect.Dis. 172:88.

Following purification of T cells, a variety of methods of geneticmodification known to those skilled in the art can be performed usingnon-viral or viral-based gene transfer vectors constructed as describedherein. For example, one such approach involves transduction of thepurified T cell population with vector-containing supernatant ofcultures derived from vector producing cells. A second approach involvesco-cultivation of an irradiated monolayer of vector-producing cells withthe purified T cells. A third approach involves a similar co-cultivationapproach; however, the purified T cells are pre-stimulated with variouscytokines and cultured 48 hours prior to the co-cultivation with theirradiated vector producing cells. Pre-stimulation prior to suchtransduction increases effective gene transfer (Nolta et al. (1992) Exp.Hematol. 20:1065). Stimulation of these cultures to proliferate alsoprovides increased cell populations for re-infusion into the patient,Subsequent to co-cultivation, T cells are collected from the vectorproducing cell monolayer, expanded, and frozen in liquid nitrogen.

Gene transfer vectors, containing one or more synthetic sequences of thepresent invention (associated with appropriate control elements fordelivery to the isolated T cells) can be assembled using known methods.

Selectable markers can also be used in the construction of gene transfervectors. For example, a marker can be used which imparts to a mammaliancell transduced with the gene transfer vector resistance to a cytotoxicagent. The cytotoxic agent can be, but is not limited to, neomycin,aminoglycoside, tetracycline, chloramphenicol, sulfonamide, actinomycin,netropsin, distamycin A, anthracycline, or pyrazinamide. For example,neomycin phosphotransferase II imparts resistance to the neomycinanalogue geneticin (G418).

The T cells can also be maintained in a medium containing at least onetype of growth factor prior to being selected. A variety of growthfactors are known in the art that sustain the growth of a particularcell type. Examples of such growth factors are cytokine mitogens such asrIL-2, IL-10, IL-12, and IL-15, which promote growth and activation oflymphocytes. Certain types of cells are stimulated by other growthfactors such as hormones, including human chorionic gonadotropin (hCG)and human growth hormone. The selection of an appropriate growth factorfor a particular cell population is readily accomplished by one of skillin the art.

For example, white blood cells such as differentiated progenitor andstem cells are stimulated by a variety of growth factors. Moreparticularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF,produced by activated TH and activated macrophages, stimulate myeloidstem cells, which then differentiate into pluripotent stem cells,granulocyte-monocyte progenitors, eosinophil progenitors, basophilprogenitors, megakaryocytes, and erythroid progenitors. Differentiationis modulated by growth factors such as GM-CSF, IL-3, IL-6, IL-1, andEPO.

Pluripotent stem cells then differentiate into lymphoid stem cells, bonemarrow stromal cells, T cell progenitors, B cell progenitors,thymocytes, TH Cells, TC cells, and B cells. This differentiation ismodulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF,M-CSF, G-CSF, IL-2, and IL-5.

Granulocyte-monocyte progenitors differentiate to monocytes,macrophages, and neutrophils. Such differentiation is modulated by thegrowth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitorsdifferentiate into eosinophils. This process is modulated by GM-CSF andIL-5.

The differentiation of basophil progenitors into mast cells andbasophils is modulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produceplatelets in response to GM-CSF, EPO, and IL-6. Erythroid progenitorcells differentiate into red blood cells in response to EPO.

Thus, during activation by the CD3-binding agent, T cells can also becontacted with a mitogen, for example a cytokine such as IL-2. Inparticularly preferred embodiments, the IL-2 is added to the populationof T cells at a concentration of about 50 to 100 μg/ml. Activation withthe CD3-binding agent can be carried out for 2 to 4 days.

Once suitably activated, the T cells are genetically modified bycontacting the same with a suitable gene transfer vector underconditions that allow for transfection of the vectors into the T cells.Genetic modification is carried out when the cell density of the T cellpopulation is between about 0.1×10⁶ and 5×10⁶, preferably between about0.5×10⁶ and 2×10⁶. A number of suitable viral and nonviral-based genetransfer vectors have been described for use herein.

After transduction, transduced cells are selected away fromnon-transduced cells using known techniques. For example, if the genetransfer vector used in the transduction includes a selectable markerthat confers resistance to a cytotoxic agent, the cells can be contactedwith the appropriate cytotoxic agent, whereby non-transduced cells canbe negatively selected away from the transduced cells. If the selectablemarker is a cell surface marker, the cells can be contacted with abinding agent specific for the particular cell surface marker, wherebythe transduced cells can be positively selected away from thepopulation. The selection step can also entail fluorescence-activatedcell sorting (FACS) techniques, such as where FACS is used to selectcells from the population containing a particular surface marker, or theselection step can entail the use of magnetically responsive particlesas retrievable supports for target cell capture and/or backgroundremoval.

More particularly, positive selection of the transduced cells can beperformed using a FACS cell sorter (e.g. a FACSVantage™ Cell Sorter,Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to sort andcollect transduced cells expressing a selectable cell surface marker.Following transduction, the cells are stained with fluorescent-labeledantibody molecules directed against the particular cell surface marker.The amount of bound antibody on each cell can be measured by passingdroplets containing the cells through the cell sorter. By imparting anelectromagnetic charge to droplets containing the stained cells, thetransduced cells can be separated from other cells. The positivelyselected cells are then harvested in sterile collection vessels. Thesecell sorting procedures are described in detail, for example, in theFACSVantage™ Training Manual, with particular reference to sections 3-11to 3-28 and 10-1 to 10-17.

Positive selection of the transduced cells can also be performed usingmagnetic separation of cells based on expression or a particular cellsurface marker. In such separation techniques, cells to be positivelyselected are first contacted with specific binding agent (e.g., anantibody or reagent the interacts specifically with the cell surfacemarker). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) that are coupled with a reagent thatbinds the specific binding agent (that has bound to the positive cells).The cell-binding agent-particle complex can then be physically separatedfrom non-labeled cells, for example using a magnetic field. When usingmagnetically responsive particles, the labeled cells can be retained ina container using a magnetic filed while the negative cells are removed.These and similar separation procedures are known to those of ordinaryskill in the art.

Expression of the vector in the selected transduced cells can beassessed by a number of assays known to those skilled in the art. Forexample, Western blot or Northern analysis can be employed depending onthe nature of the inserted nucleotide sequence of interest. Onceexpression has been established and the transformed T cells have beentested for the presence of the selected synthetic sequence, they areready for infusion into a patient via the peripheral blood stream.

The invention includes a kit for genetic modification of an ex vivopopulation of primary mammalian cells. The kit typically contains a genetransfer vector coding for at least one selectable marker and at leastone synthetic sequence (e.g., expression cassette) contained in one ormore containers, ancillary reagents or hardware, and instructions foruse of the kit.

6.3 Further Delivery Regimes

Any of the polynucleotides (e.g., expression cassettes) or polypeptidesdescribed herein (delivered by any of the methods described above) canalso be used in combination with other DNA delivery systems and/orprotein delivery systems. Non-limiting examples includeco-administration of these molecules, for example, in prime-boostmethods where one or more molecules are delivered in a “priming” stepand, subsequently, one or more molecules are delivered in a “boosting”step.

In certain embodiments, the delivery of one or more nucleicacid-containing compositions is followed by delivery of one or morenucleic acid-containing compositions and/or one or morepolypeptide-containing compositions (e.g., polypeptides comprising HIVantigens). In other embodiments, the delivery of one or more nucleicacid-containing compositions is preceded by delivery of one or morepolypeptide-containing compositions (e.g., polypeptides comprising HIVantigens) and/or one or more nucleic acid-containing compositions. Instill other embodiments, multiple nucleic acid “primes” (of the same ordifferent nucleic acid molecules) can be followed by multiplepolypeptide “boosts” (of the same or different polypeptides) and/ormultiple polynucleotide “boosts” (of the same or differentpolynucleotides). Other examples include multiple nucleic acidadministrations and multiple polypeptide administrations.

In any method involving co-administration, the various compositions canbe delivered in any order. Thus, in embodiments including delivery ofmultiple different compositions or molecules, the nucleic acids need notbe all delivered before the polypeptides. For example, the priming stepmay include delivery of one or more polypeptides and the boostingcomprises delivery of one or more nucleic acids and/or one morepolypeptides. Multiple polypeptide administrations can be followed bymultiple nucleic acid administrations or polypeptide and nucleic acidadministrations can be performed in any order. In any of the embodimentsdescribed herein, the nucleic acid molecules can encode all, some ornone of the polypeptides. Thus, one or more or the nucleic acidmolecules (e.g., expression cassettes) described herein and/or one ormore of the polypeptides described herein can be co-administered in anyorder and via any administration routes. Therefore, any combination ofpolynucleotides and/or polypeptides described herein can be used togenerate elicit an immune reaction.

7 Compositions

Recombinant vectors carrying a synthetic sequences of the presentinvention are typically formulated into compositions for delivery to thevertebrate subject. These compositions may either be prophylactic (toprevent infection) or therapeutic (to treat disease after infection) andmay include one or more of the following molecules: polynucleotides,polypeptides, other small molecules and/or other macromolecules. Thecompositions will comprise a “therapeutically effective amount” of thegene of interest such that an amount of the antigen can be produced invivo so that an immune response is generated in the individual to whichit is administered. The exact amount necessary will vary depending onthe subject being treated; the age and general condition of the subjectto be treated; the capacity of the subject's immune system to synthesizeantibodies; the degree of protection desired; the severity of thecondition being treated; the particular antigen selected and its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present in such vehicles.Certain facilitators of nucleic acid uptake and/or expression can alsobe included in the compositions or co-administered, such as, but notlimited to, bupivacaine, cardiotoxin and sucrose.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Examples of particulate carriers include those derived frompolymethyl methacrylate polymers, as well as microparticles derived frompoly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g.,Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., JMicroencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993. Such carriers are well known to those of ordinaryskill in the art. Additionally, these carriers may function asimmunostimulating agents (“adjuvants”). Furthermore, the antigen may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc., as well as toxins derived from E. coli.

Compositions of the invention may be administered in conjunction withother immunoregulatory agents. In particular, the compositions willusually include an adjuvant. Adjuvants for use with the inventioninclude, but are not limited to, one or more of the following set forthbelow.

Adjuvants may also be used to enhance the effectiveness of thecompositions. Such adjuvants include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3)saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) oligonucleotides or polymeric moleculesencoding immtmostimulatory CpG motifs (a sequence containing anunmethylated cytosine followed by guanosine and linked by a phosphatebond) (Davis, H. L., et al., J. Immunology 160:870-876, 1998; Sato, Y.et al., Science 273:352-354, 1996) or complexes ofantigens/oligonucleotides {Polymeric molecules include double and singlestranded RNA and DNA, and backbone modifications thereof, for example,methylphosphonate linkages; or (7) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT), particularly LT-K63 (wherelysine is substituted for the wild-type amino acid at position 63)LT-R72 (where arginine is substituted for the wild-type amino acid atposition 72), CT-S109 (where serine is substituted for the wild-typeamino acid at position 109), and PT-K9/G129 (where lysine is substitutedfor the wild-type amino acid at position 9 and glycine substituted atposition 129) (see, e.g., International Publication Nos. WO93/13202 andWO92/19265); and (8) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition. Further, suchpolymeric molecules include alternative polymer backbone structures suchas, but not limited to, polyvinyl backbones (Pitha, Biochem BiophysActa, 204:39, 1970a; Pitha, Biopolymers, 9:965, 1970b), and morpholinobackbones (Summerton, J., et al., U.S. Pat. No. 5,142,047, issued Aug.25, 1992; Summerton, J., et al., U.S. Pat. No. 5,185,444 issued Feb. 9,1993). A variety of other charged and uncharged polynucleotide analogshave been reported. Numerous backbone modifications are known in theart, including, but not limited to, uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, and carbamates) andcharged linkages (e.g., phosphorothioates and phosphorodithioates).

Other Adjuvants Include, but are not Limited to:

(1) Virosomes and Virus Like Particles (VLPs): These structuresgenerally contain one or more proteins from a virus optionally combinedor formulated with a phospholipid. They are generally non-pathogenic,non-replicating and generally do not contain any of the native viralgenome. The viral proteins may be recombinantly produced or isolatedfrom whole viruses. These viral proteins suitable for use in virosomesor VLPs include proteins derived from influenza virus (such as HA orNA), Hepatitis B virus (such as core or capsid proteins), Hepatitis Evirus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Diseasevirus, Retrovirus, Norwalk virus, human Papilloma virus, HIV,RNA-phages, Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205phage, and Ty (such as retrotransposon Ty protein p1). VLPs arediscussed further in WO03/024480, WO03/024481, and Niikura et al.,Virology (2002) 293:273-280; Lenz et al., Journal of Immunology (2001)5246-5355; Pinto, et al., Journal of Infectious Diseases (2003)188:327-338; and Gerber et al., Journal of Virology (2001)75(10):4752-4760. Virosomes are discussed further in, for example, Glucket al., “New Technology Platforms in the Development of Vaccines for theFuture”, Vaccine (2002) 20:B10-B16. Immunopotentiating reconstitutedinfluenza virosomes (IRIV) are used as the subunit antigen deliverysystem in the intranasal trivalent IFLEXAL™ product {Mischler & Metcalfe(2002) Vaccine 20 Suppl 5:B17-23} and the INFLUVAC PLUS™ product.

(2) Bacterial or Microbial Derivatives Such as:

(a) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3 dMPL). 3 dMPL is a mixture of 3 De-O-acylated monophosphoryllipid A with 4, or 6 acylated chains. A preferred “small particle” formof 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454.Such “small particles” of 3dMPL are small enough to be sterile filteredthrough a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives e.g. RC-529. See Johnson et al.(1999) Bioorg Med Chem Lett 9:2273-2278.

(b) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in Meraldi et al.,“OM-174, a New Adjuvant with a Potential for Human Use, Induces aProtective Response with Administered with the Synthetic C-TerminalFragment 242-310 from the circumsporozoite protein of Plasmodiumberghei”, Vaccine (2003) 21:2485-2491; and Pajak, et al., “The AdjuvantOM-174 induces both the migration and maturation of murine dendriticcells in vivo”, Vaccine (2003) 21:836-842.

(c) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants includenucleotide sequences containing a CpG motif (a sequence containing anunmethylated cytosine followed by guanosine and linked by a phosphatebond). Bacterial double stranded RNA or oligonucleotides containingpalindromic or poly(dG) sequences have also been shown to beimmunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. Optionally, the guanosine may be replaced with ananalog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al.,“Divergent synthetic nucleotide motif recognition pattern: design anddevelopment of potent immunomodulatory oligodeoxyribonucleotide agentswith distinct cytokine induction profiles”, Nucleic Acids Research(2003) 31(9): 2393-2400; WO02/26757 and WO99/62923 for examples ofpossible analog substitutions. The adjuvant effect of CpGoligonucleotides is further discussed in Krieg, “CpG motifs: the activeingredient in bacterial extracts?”, Nature Medicine (2003) 9(7):831-835; McCluskie, et al., “Parenteral and mucosal prime-boostimmunization strategies in mice with hepatitis B surface antigen and CpGDNA”, FEMS Immunology and Medical Microbiology (2002) 32:179-185;WO98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S.Pat. No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT. See Kandimalla, et al., “Toll-like receptor 9: modulation ofrecognition and cytokine induction by novel synthetic CpG DNAs”,Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpGsequence may be specific for inducing a Th1 immune response, such as aCpG-A ODN, or it may be more specific for inducing a B cell response,such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, etal., “CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Productionis Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha”, J.Immunol. (2003) 170(8):4061-4068; Krieg, “From A to Z on CpG”, TRENDS inImmunology (2002) 23(2): 64-65 and WO01/95935. Preferably, the CpG is aCpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, Kandimalla, et al., “Secondary structures in CpGoligonucleotides affect immunostimulatory activity”, BBRC (2003)306:948-953; Kandimalla, et al., “Toll-like receptor 9: modulation ofrecognition and cytokine induction by novel synthetic GpG DNAs”,Biochemical Society Transactions (2003) 31 (part 3):664-658; Bhagat etal., “CpG penta- and hexadeoxyribonucleotides as potent immunomodulatoryagents” BBRC (2003) 300:853-861 and WO03/035836.

(3) Bioadhesives and Mucoadhesives: Suitable Bioadhesives IncludeEsterified hyaluronic acid microspheres (Singh et al. (2001) J. Cont.Rele. 70:267-276) or mucoadhesives such as cross-linked derivatives ofpolyacrylic acid, polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention. E.g. WO99/27960.

(4) Microparticles: Microparticles (i.e. a particle of ˜100 nm to ˜15 μmin diameter, more preferably ˜200 nm to ˜30 μn in diameter, and mostpreferably ˜500 mm to 10 μm in diameter) formed from materials that arebiodegradable and non-toxic (e.g. a poly(α-hydroxy acid), apolyhydroxybutyric acid, a polyorthoester, a polyanhydride, apolycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred,optionally treated to have a negatively-charged surface (e.g. with SDS)or a positively-charged surface (e.g. with a cationic detergent, such asCTAB).

(5) Liposome formulations: Examples of liposome formulations suitablefor use as adjuvants are described in U.S. Pat. No. 6,090,406, U.S. Pat.No. 5,916,588, and EP 0 626 169.

(6) Polyoxyethylene ethers and polyoxyethylene esters: Polyoxyethyleneethers and polyoxyethylene esters are described in, for example,WO99/52549. Such formulations further include polyoxyethylene sorbitanester surfactants in combination with an octoxynol (WOO 1/21207) as wellas polyoxyethylene alkyl ethers or ester surfactants in combination withat least one additional non-ionic surfactant such as an octoxynol(WO01/21152). Preferred polyoxyethylene ethers are selected from thefollowing group: polyoxyethylene-9-lauryl ether (laureth 9),polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether,polyoxyethylene4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

(7) Polyphosphazene (PCPP): PCPP formulations are described, forexample, in Andrianov et al., “Preparation of hydrogel microspheres bycoacervation of aqueous polyphophazene solutions”, Biomaterials (1998)19 (1-3):109-115 and Payne et al., “Protein Release from PolyphosphazeneMatrices”, Adv. Drug. Delivery Review (1998) 31(3):185-196.

(8) Imidazoquinoline Compounds: Examples of imidazoquinoline compoundssuitable for use as adjuvants include Imiquimod and its analogues,described further in Stanley, Clin Exp Demnatol (2002) 27(7):571-577;Jones, Curr Opin Investig Drugs (2003) 4(2):214-218; and U.S. Pat. Nos.4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784,5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612.

(9) Thiosemicarbazone Compounds: Examples of thiosemicarbazonecompounds, as well as methods of formulating, manufacturing, andscreening for compounds all suitable for use as adjuvants include thosedescribed in WO04/60308.

(10) Tryptanthrin Compounds: Examples of tryptanthrin compounds, as wellas methods of formulating, manufacturing, and screening for compoundsall suitable for use as adjuvants in the invention include thosedescribed in WO04/64759.

(11) Human Immunomodulators: Human immunomodulators suitable for use asadjuvants include cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

Compositions of the invention may be formulated with one or more of theadjuvants identified above. For example, the following adjuvantcompositions may be used in the invention:

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., using one or more of the methodsdescribed above) or, alternatively, delivered ex vivo, to cells derivedfrom the subject, using methods such as those described above. Forexample, methods for the ex vivo delivery and reimplantation oftransformed cells into a subject are known in the art and can include,e.g., dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, lipofectamine and LT-1 mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) (with or without the corresponding antigen) inliposomes, and direct microinjection of the DNA into nuclei.

The amount of DNA administered to the subject may vary depending on theantigens and/or delivery protocol. Thus, in certain embodiments, theamount of DNA used per administration (e.g., immunization) may vary fromnanogram to microgram to milligram amounts of DNA, for example, asdescribed in the Examples where the dose given is between about 2nanograms to 20 micrograms or between about 2 nanograms and 10milligrams. Further, as described below and shown in the Examples,multiple administrations of DNA (and/or protein) are contemplated.

Dosage treatment may be a single dose schedule or a multiple doseschedule. Administration of nucleic acids may also be combined withadministration of peptides or other substances.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Generation of Synthetic Sequences

The synthetic sequences were generated from sequences obtained fromHIVSF2 isolates. These sequences were manipulated to maximize expressionof their gene products.

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes. The HIV codon usage reflects a highcontent of the nucleotides A or T of the codon-triplet. The effect ofthe HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The coding sequences were modified to be comparable to codon usagefound in highly expressed human genes.

Certain Pol-encoding sequences were also modified in certain cases. Forexample, the protease (Prot) region of Pol was modified in certainsequences to that the protease activity was attenuated (constructsdesignated “pATT”) or such that protease activity was inactivated(constructs designated “pIna”).

The synthetic coding sequences were assembled by methods known in theart, for example by companies such as the Midland Certified ReagentCompany (Midland, Tex.) or RetroGen (San Diego, Calif.) and cloned intothe following eukaryotic expression vectors: pCMVlink or pCMVKm2. For adescription of construction of these vectors, see, for example, WO00/39302. Exemplary synthetic sequences are shown in FIGS. 1-2.

Example 2 Expression Assays for the Synthetic Coding Sequences

The synthetic sequences are cloned into expression vectors in varyingorientations.

Expression efficiencies for various vectors carrying the single ormultiple synthetic sequences in various orientations are evaluated asfollows. Cells from several mammalian cell lines (293, RD, COS-7, andCHO; all obtained from the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209) are transfected with 2μg of DNA in transfection reagent LT1 (PanVera Corporation, 545 ScienceDr., Madison, Wis.). The cells are incubated for 5 hours in reducedserum medium (Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The medium isthen replaced with normal medium as follows: 293 cells, 1 M, 10% fetalcalf serum, 2% glutamine (BioWhittaker, Walkersville, Minn.); RD andCOS-7 cells, D-MEM, 10% fetal calf serum, 2% glutamine (Opti-MEM,Gibco-BRL, Gaithersburg, Md.); and CHO cells, Ham's F-12, 10% fetal calfserum, 2% glutamine (Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The cellsare incubated for either 48 or 60 hours. Cell lysates are collected asdescribed below in Example 3. Supernatants are harvested and filteredthrough 0.45 μm syringe filters. Supernatants are evaluated using theusing 96-well plates coated with a rabbit polyclonal IgG directedagainst the appropriate HIV polypeptide (e.g., p24, p55, etc.).Biotinylated antibodies against HIV recognize the bound antigen.Conjugated strepavidin-horseradish peroxidase reacts with the biotin.Color develops from the reaction of peroxidase with TMB substrate. Thereaction is terminated by addition of 4N H₂SO4. The intensity of thecolor is directly proportional to the amount of HIV antigen in a sample.

Example 3 Western Blot Analysis of Expression

Human 293 cells were transfected as described in Example 2 with theconstructs (all pCMV-based) containing the following sequences:synthetic Gag-encoding sequences; in-frame synthetic Gag- and Pol(inactivated)-encoding sequences; in-frame synthetic sequences, ordered5′ to 3′, Gag-, Pol (inactivated)-, Tat-, Rev-, Nef-encoding sequences,in-frame synthetic sequences, ordered 5′ to 3′, Tat-, Rev-, Nef-, Gag-,Pol (inactivated)-encoding sequences; in-frame synthetic Gag- and Pol(attenuated)-encoding sequences; in-frame synthetic sequences, ordered5′ to 3′, Gag-, Pol (attenuated)-, Tat-, Rev-, Nef-encoding sequences;and in-frame synthetic sequences, ordered 5′ to 3′, Tat-, Rev-, Nef-,Gag-, Pol (attenuated)-encoding sequences.

Cells were cultivated for 48 or 72 hours post-transfection. Cell lysateswere prepared as follows. The cells were washed once withphosphate-buffered saline, lysed with detergent [1% NP40 (Sigma ChemicalCo., St. Louis, Mo.) in 0.1 M Tris-HCl, pH 7.5], and the lysatetransferred into fresh tubes. Supernatants were prepared as previouslydescribed and 20 μl of supernatant loaded in reduced condition into aTris-bis gel and run with MES running buffer.

SDS-polyacrylamide gels (pre-cast 8-16%; Novex, San Diego, Calif.) wereloaded with 20 μl of supernatant or 10 μl of cell lysate. A proteinstandard was also loaded (5 μl, broad size range standard; BioRadLaboratories, Hercules, Calif.). Electrophoresis was carried out and theproteins were transferred using a BioRad Transfer Chamber (BioRadLaboratories, Hercules, Calif.) to Immobilon P membranes (MilliporeCorp., Bedford, Mass.) using the transfer buffer recommended by themanufacturer (Millipore), where the transfer is performed at 100 voltsfor 90 minutes. The membranes are exposed to HIV-1-positive humanpatient serum (1:400 dilution) and anti-β actin (dilution 1:5000) for 1hr at room temperature. Anti-mouse and anti-human antibodies (1:30000dilution) were used for detection using Odyssey™ Antibody Detection Kits(Li-Cor Biosciences, Lincoln, Nebr.), following the manufacturer'sinstructions.

In the experiments shown in FIGS. 3 and 4, synthetic HIV polypeptideencoding sequences (e.g., in expression cassettes) in which Tat, revand/or nef were positioned 5′ to Gag- and Pol-encoding sequencesexhibited increased production of their protein products, relative towild-type and to synthetic sequences in which Tat, rev and/or nef werepositioned 3′ to Gag- and Pol-encoding sequences. (FIG. 3, especiallycomparing lane 3 to lane 4 and lane 6 to lane 7) and supernatants (FIG.4, especially comparing lane 3 to lane 4 and lane 6 to lane 7).

FIGS. 5 and 6 show similar experiments in which increased expression ofGag and/or pol from constructs in which Tat, rev and/or nef werepositioned 5′ to Gag- and Pol-encoding sequences was not observed.

Example 4 In Vivo Immunogenicity of Synthetic HIV Expression Cassettes

A. Immunization

To evaluate the immunogenicity of the synthetic HIV expressioncassettes, mouse studies were performed as follows. The plasmid DNA,e.g., pCMVKM2 carrying an expression cassette comprising a syntheticsequence of the present invention, was diluted to the following finalconcentrations in a total injection volume of 100 μl: 20 μg, 2 μg, 0.2μg, and 0.02 μg. In particular 18 groups of 4 animals and 1 group of 3animals were immunized as shown in the following Table. In all Groups,the route of administration was intramuscular injection into thetibialis anterior.

Grp. Anim./ Animal # of Total Total Vol/ Sites/ No. grp No.Immunizations Immunogen Dose Vol. Site Animal 1 4 1-4 2 pCMV-TRN 20 ug100 μl 50 μl 2 2 4 5-8 2 pCMV-TRN 2 ug 100 μl 50 μl 2 3 4  9-12 2pCMV-TRN 0.2 ug 100 μl 50 μl 2 4 4 13-16 2 pCMV-gagCpollna 20 ug 100 μl50 μl 2 5 4 17-20 2 pCMV-gagCpollna 2 ug 100 μl 50 μl 2 6 4 21-24 2pCMV-gagCpollna 0.2 ug 100 μl 50 μl 2 7 4 25-28 2 pCMV-TRN + gagCpollna20 ug 100 μl 50 μl 2 8 4 29-32 2 pCMV-TRN + gagCpollna 2 ug 100 μl 50 μl2 9 4 33-36 2 pCMV-TRN + gagCpollna 0.2 ug 100 μl 50 μl 2 10 4 37-40 2pCMV-TRNgagCpollna 20 ug 100 μl 50 μl 2 11 4 41-44 2 pCMV-TRNgagCpollna2 ug 100 μl 50 μl 2 12 4 45-48 2 pCMV-TRNgagCpollna 0.2 ug 100 μl 50 μl2 13 4 49-52 2 pCMV-gagCpollnaTRN 20 ug 100 μl 50 μl 2 14 4 53-56 2pCMV-gagCpollnaTRN 2 ug 100 μl 50 μl 2 15 4 57-60 2 pCMV-gagCpollnaTRN0.2 ug 100 μl 50 μl 2 16 4 61-64 2 pCMV-Tat + gagCpollna 20 ug 100 μl 50μl 2 17 4 65-68 2 pCMV-Tat + gagCpollna 2 ug 100 μl 50 μl 2 18 4 69-72 2pCMV-Tat + gagCpollna 0.2 ug 100 μl 50 μl 2 19 3 73-75 2 naive — — — —

On the day of injection one 100 μl vial of plasmid was used, with 50 μlinjected IM into the tibialis anterior (TA) muscle of both legs (50 μlper leg=100 μl total per animal), according the following immunizationschedule.

GROUPS 1-18 GROUPS 1-18 Immunization: 1 2 Weeks: 0 4 Group 1 pCMV-TRNpCMV-TRN 2 ″ ″ 3 ″ ″ 4 pCMV-gagCpollna pCMV-gagCpollna 5 6 ″ ″ 7pCMV-TRN + gagCpollna pCMV-TRN + gagCpollna 8 ″ ″ 9 10pCMV-TRNgagCpollna pCMV-TRNgagCpollna 11 ″ ″ 12 ″ ″ 13 gagCpollnaTRNgagCpollnaTRN 14 ″ ″ 15 ″ ″ 16 pCMV-Tat + gagCpollna pCMV-Tat +gagCpollna 17 18 ″ ″

To overcome possible negative dilution effects of the diluted DNA, thetotal DNA concentration in each sample was brought up to 20 μg using thevector (pCMVKM2) alone. Alphavirus vectors (e.g., Sindbis) comprisingthe same sequences are also prepared. Controls (wild type orunicistronic synthetic sequences) were handled in the same manner.

To test immune responses, animals were bled according to the followingschedule:

All 19 Groups Bleed: 1 Week: 6 Sample: Clotted Bld. for Serum Volume:200 μl Method: TB/SOB. Hurmoral Immune Response

Humoral immune responses were checked with a suitable anti-HIV antibodyELISAs (enzyme-linked immunosorbent assays). Briefly, sera fromimmunized mice were screened for antibodies directed against anappropriate HIV protein (e.g., HIV p55 for Gag). ELISA microtiter plateswere coated with 0.2 μg of HIV protein per well overnight and washedfour times; subsequently, blocking was done with PBS-0.2% Tween (Sigma)for 2 hours. After removal of the blocking solution, 100 μl of dilutedmouse serum was added. Sera are tested at 1/25 dilutions and by serial3-fold dilutions, thereafter. Microtiter plates were washed four timesand incubated with a secondary, peroxidase-coupled anti-mouse IgGantibody (Pierce, Rockford, Ill.). ELISA plates were washed and 100 μlof 3, 3′, 5,5′-tetramethyl benzidine (TMB; Pierce) was added per well.The optical density of each well was measured after 15 minutes. Thetiters reported are the reciprocal of the dilution of serum that gave ahalf-maximum optical density (O.D.). Results are shown in FIG. 7.

C. Cellular Immune Response

Intracellular cytokine staining (ICS) for Gag and Pol-specificIFN-g-producing CD8+ lymphocytes was performed essentially as describedin zur Megede et al. (2003) J. Virol. 77(11):6197-6207 and zur Megede etal. (2000) J. Virol. 74:2628-2635. Briefly, spleens were harvested twoweeks post-second DNA immunization or five days post recombinantvaccinia challenge and single cell suspensions were prepared. 1×10⁶nucleated spleen cells were cultured in duplicate at 37° C. in thepresence or absence of 10 μg/ml p7g peptide for Gag. Unstimulated cellsplus spleen cells from naive mice were used as background and negativecontrols. The background values were generally very low, between 0.01and 0.1% of IFN-g secreting CD8+ cells.

After 5 hours, cells were washed, incubated with anti-CD16/32(Pharmingen, San Diego, Calif.) to block Fcg receptors, and fixed in 1%(w/v) paraformaldehyde and stored overnight at 4° C. The following daycells were stained with FITC-conjugated CD8 mAb (Pharmingen), washed,treated with 0.5% (w/v) Saponin (Sigma) and then incubated withphycoerythrin (PE)-conjugated mouse IFN-g mAb (Pharmingen) in thepresence of 0.1% (w/v) saponin. Cells were then washed and analyzed on aFACSCalibur flow cytometer (Becton-Dickinson Immunocytometry Systems,San Jose, Calif.). Results are shown in FIG. 8.

Cellular immune responses are also tested by 1) CTL bulk culture and⁵¹Cr-release, 2) lymphoproliferation, and 3) ELISPOT. (See, also,Cherpelis et al. (2001) Immunol. Lett. 79:47-55 (describing LPA assays);and Vajdy et al. (2001) J. Infect Dis 15; 184(12):1613-1616 (describingELISPOT)).

Example 5 In Vivo Immunogenicity of Synthetic HIV Expression Cassettes

A. General Immunization Methods

To evaluate the immunogenicity of the synthetic HIV expressioncassettes, studies using guinea pigs, rabbits, mice, rhesus macaques andbaboons are performed. The studies are typically structured as follows:DNA immunization alone (single or multiple); DNA immunization followedby protein immunization (boost); DNA immunization followed by Sindbisparticle immunization; immunization by Sindbis particles alone.

B. Guinea Pigs

Experiments may be performed using guinea pigs as follows. Groupscomprising six guinea pigs each are immunized intramuscularly ormucosally at 0, 4, and 12 weeks with plasmid DNAs encoding expressioncassettes comprising one or more the sequences described herein. Theanimals are subsequently boosted at approximately 18 weeks with a singledose (intramuscular, intradermally or mucosally) of the HIV proteinencoded by the sequencers) of the plasmid boost and/or other HIVproteins. Antibody titers (geometric mean titers) are measured at twoweeks following the third DNA immunization and at two weeks after theprotein boost. These results are used to demonstrate the usefulness ofthe synthetic constructs to generate immune responses, as well as, theadvantage of providing a protein boost to enhance the immune responsefollowing DNA immunization.

C. Rabbits

Experiments may be performed using rabbits as follows. Rabbits areimmunized intramuscularly, mucosally, or intradermally (using a Biojectneedless syringe) with plasmid DNAs encoding the HIV proteins describedherein. The nucleic acid immunizations are followed by protein boostingafter the initial immunization. Typically, constructs comprising thesynthetic HIV-polypeptide-encoding polynucleotides of the presentinvention are highly immunogenic and generate substantial antigenbinding antibody responses after only 2 immunizations in rabbits.

D. Baboons

Four baboons are immunized 3 times (weeks 0, 4 and 8) bilaterally,intramuscular into the quadriceps or mucosally using the gene deliveryvehicles described herein.

Lymphoproliferative responses to are observed in baboons two weekspost-fourth immunization (at week 14), and enhanced substantiallypost-boosting with HIV-polypeptide (at week 44 and 76). Suchproliferation results are indicative of induction of T-helper cellfunctions.

The animals are also bled two weeks after each immunization and an HIVantibody ELISA is performed with isolated plasma. The ELISA is performedessentially as described above except the second antibody-conjugate isan anti-human IgG, g-chain specific, peroxidase conjugate (SigmaChemical Co., St. Louis, Md. 63178) used at a dilution of 1:500. Fiftyμg/ml yeast extract may be added to the dilutions of plasma samples andantibody conjugate to reduce non-specific background due to preexistingyeast antibodies in the baboons.

E. Rhesus Macagues

The improved immunogenic potency of the synthetic, codon-modifiedHIV-polypeptide encoding polynucleotides of the present invention, whenconstructed into expression plasmids may be confirmed in rhesusmacaques. Typically, the macaques have detectable HIV-specific CTL aftertwo or three 1 mg doses of modified HIV polynucleotide. In sum, theseresults demonstrate that the synthetic HIV DNA is immunogenic innon-human primates. Neutralizing antibodies may also detected.

F. Immune Responses

Cellular and humoral immune responses were evaluated in mice(essentially as described in Example 4) for the following constructs:TatRefNef (TRN), GagcPolIna, GagcPolTatRefNef (gCpInaTRN3),TatRefNefGagcPolIna (5TRNgCpIna), GagcPolatt (gCpAtt),GagcPolAttTatRefNef (gCpAttTRN3), and TatRevNefGagcPolAtt (5TRNgCpIna).

As summarized in Examples 2 and 3, in vitro expression certain datashowed increased expression of Gag-Pol proteins (e.g., p24 and, p55Gagand p66RT) using polycistronic constructs in which Tat, Ref and Nef werepositioned 5′ to Gag and Pol. (FIGS. 3 and 4). Other data showed noincreased expression based on the relative positions of the nucleotides(FIGS. 5 and 6).

Previous studies have shown that the immune response in mammaliansubjects correlates with the relative levels of protein expression. See,e.g., 6,602,705 and International Publications WO 00/39303, WO 00/39302,WO 00/39304, WO 02/04493. Therefore, it is expected that 5TRNgCpIna and5TRNgCpAtt will generate robust immune responses in vivo.

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of theinvention.

1. A method of generating an immune response in a subject, the methodcomprising the step of administering to the subject a vector underconditions such that the sequences encoding the HIV polypeptides areexpressed, wherein the vector comprises a synthetic polynucleotidecomprising polynucleotide sequences encoding a polyprotein comprisingHIV Gag, Pol, Tat, Rev and Nef polypeptides, wherein the polynucleotidesequence is operably linked to control elements, wherein thepolynucleotide sequences encoding Gag and Pol polypeptides are located3′ to polynucleotide sequences encoding the Tat, Rev, and Nefpolypeptides, wherein expression of the polynucleotide sequence isincreased relative to expression of a native polynucleotide sequenceencoding the polypeptides.
 2. The method of claim 1, further comprisingthe step of administering one or more additional polypeptides to thesubject.
 3. The method of claim 2, wherein the additional polypeptidesare HIV polypeptides.
 4. The method of claim 3, where in the additionalpolypeptides are encoded by the vector.
 5. The method of claim 2,wherein the vector and polypeptides are administered sequentially. 6.The method of claim 2, wherein the vector and polypeptides areadministered concurrently.
 7. The method of claim 1, wherein the vectoris delivered using a particulate carrier.
 8. The method of claim 1,wherein the vector is coated on a gold or tungsten particle and thecoated particle is delivered to the subject using a gene gun.
 9. Themethod of claim 1, wherein the vector is encapsulated in a liposomepreparation.
 10. The method of claim 1, wherein the subject is a mammal.11. The method of claim 10, wherein the mammal is a human.
 12. Themethod of claim 1, where the immune response is a humoral immuneresponse.
 13. The method of claim 1, where the immune response is acellular immune response.
 14. The method of claim 1, wherein the vectoris administered intramuscularly, intramucosally, intranasally,subcutaneously, intradermally, transdermally, intravaginally,intrarectally, orally or intravenously.
 15. A method of claim 1, furthercomprising the step of co-administering an additional immunogenicmolecule.
 16. The method of claim 15, wherein the additional moleculecomprises one or more gene delivery vehicles encoding one or more HIVproteins.